Method and microorganisms for the production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (hmbpa)

ABSTRACT

The present invention features methods of producting 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”) and α-hydroxyisovalerate (“α-HIV”) utilizing microorganisms having modified pantothenate biosynthetic enzyme activities. Recombinant microorganisms and conditions for culturing same are also featured. Also featured are compositions including HMBPA and compositions including α-HIV.

RELATED APPLICATIONS

[0001] The present invention claims the benefit of prior-filedprovisional Patent Application Serial No. 60/263,053, filed Jan. 19,2001 (pending). The present invention is also related to U.S. patentapplication Ser. No. 09/667,569, filed Sep. 21, 2000 (pending), which isa continuation-in-part of U.S. patent application Ser. No. 09/400,494,filed Sep. 21, 1999 (abandoned). U.S. patent application Ser. No.09/667,569 also claims the benefit of prior-filed provisional PatentApplication Serial No. 60/210,072, filed Jun. 7, 2000, provisionalPatent Application Serial No. 60/221,836, filed Jul. 28, 2000, andprovisional Patent Application Serial No. 60/227,860, filed Aug. 24,2000. The entire content of each of the above-referenced applications isincorporated herein by this reference.

BACKGROUND OF THE INVENTION

[0002] Conventional means of synthesizing chemical compounds is viasynthesis from bulk chemicals, a process which is limited by factorssuch as substrate availability and/or cost, difficulty in resolvingcomplex mixtures of products, complexities in synthesizing largequantities of compounds in purified form, and difficulty in producingchiral compounds. Accordingly, researchers have recently looked tobacterial or microbial systems that express enzymes useful for variousbiosynthetic processes, for example, in the synthesis of pharmaceuticalcompounds, research reagents, nutriceuticals, vitamins, nutritionalsupplements, antibiotic compounds and the like. In particular,bioconversion processes have been evaluated as a means of favoringproduction of preferred compounds and recently methods of directmicrobial synthesis have been the focus of much research in the areas ofpharmaceuticals and agriculture.

SUMMARY OF THE INVENTION

[0003] The present invention relates to a processes for the directmicrobial synthesis of [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionicacid or 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”),referred to interchangeably herein as “β-alanine2-(R)-hydroxyisolvalerate”, “β-alanine 2-hydroxyisolvalerate”,“β-alanyl-α-hydroxyisovalarate”,N-(2-hydroxy-3-methyl-1-oxobutyl)-β-alanine (“HMOBA”) and/or“fantothenate”. In particular, it has been discovered that inmicroorganisms engineered to overexpress certain enzymes conventionallyassociated with pantothenate and/or isoleucine-valine (ilv)biosynthesis, an alternative biosynthetic pathway is present thatcompetes for key precursors of pantothenate biosynthesis, namelyα-ketoisovalerate (α-KIV) and β-alanine. α-KIV is converted toα-hydroxyisovalerate (α-HIV) catalyzed by various reductase enzymes andα-HIV is subsequently condensed with β-alanine to produce HMBPA.

[0004] In one embodiment, the invention features a process for theproduction of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA)that includes culturing a microorganism having increased keto reductaseactivity or increased pantothenate synthetase activity in the presenceof excess α-ketoisovalerate and excess β-alanine, such that HMBPA isproduced. In another embodiment, the invention features a process forthe production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid(HMBPA) that includes culturing a microorganism having increased ketoreductase activity and increased pantothenate synthetase activity in thepresence of excess α-ketoisovalerate and excess β-alanine, such thatHMBPA is produced. In one embodiment, the microorganism has a modifiedpanE gene, for example, a modified panel gene and/or a modified panE2gene (e.g., the panE gene is overexpressed, deregulated or present inmultiple copies). In another embodiment, the microorganism has amodified panC gene (e.g., the panC gene is overexpressed, deregulated orpresent in multiple copies). In another embodiment, the microorganismfurther has increased acetohydroxyacid isomeroreductase activity. Inanother embodiment, the microorganism is cultured under conditions ofincreased acetohydroxyacid isomeroreductase activity in the presence ofexcess α-ketoisovalerate and excess β-alanine, such that HMBPA isproduced. In yet another embodiment, the microorganism comprises amodified ilvC gene (e.g., the ilvC gene is overexpressed, deregulated orpresent in multiple copies). In yet another embodiment, themicroorganism further has reduced ketopantoate hydroxymethyltransferaseactivity (e.g., has a modified panB gene, for example a panB gene thathas been deleted.

[0005] In another aspect, the invention features a process for theproduction of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA)that includes culturing a microorganism having reduced ketopantoatehydroxymethyltransferase activity in the presence of excessα-ketoisovalerate and excess β-alanine, such that HMBPA is produced. Inanother aspect, the invention features a method for enhancing productionof 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) relativeto pantothenate that includes culturing a recombinant microorganismunder conditions such that the HMBPA production is enhanced relative topantothenate production. In another aspect, the invention features aprocess for the production of 2-hydroxyisovaleric acid (α-HIV) thatincludes culturing a microorganism which overexpresses PanE1 or PanE2and which further has reduced PanC or PanD activity under conditionssuch that α-HIV is produced. In another aspect, the invention features aprocess for the production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) that includesculturing a recombinant microorganism having decreased expression oractivity of serA or glyA under conditions such that HMBPA is produced.In another aspect, the invention features a process for the productionof 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) thatincludes culturing a recombinant microorganism having decreasedexpression or activity of serA and glyA under conditions such that HMBPAis produced. Conditions for culturing the above described microorganismsinclude, for example, conditions of increased steady state glucose,conditions of decreased steady state dissolved oxygen, and/or culturedunder conditions of decreased serine. Products produced according to theabove described processes and/or methods are also featured. Alsofeatured are recombinant microorganisms utilized in the above-describedmethods.

[0006] Compounds produced according to the methodologies of the presentinvention have a variety of uses. For example, HMBPA can be used tosynthesize inhibitors of HMG CoA Reductase (II) (Gordon et al. Bio. Med.Chem. Lett. 1(3):161 (1991). Inhibitors of HMG CoA Reductase (II) havebeen studied for use as in the treatment of hypercholesterolaemia andcoronary atherosclerosis progression. Inhibitors of HMG CoA Reductasealso have been used to reduce risk of cardiovascular events in patientsat risk. Moreover, the HMBPA precursor 2-hydroxyisovalerate (α-HIV) hasbeen demonstrated to have nutriceutical properties, for example, in theprevention of aging of the skin. In particular, α-hydroxy acids, such asα-HIV (or 2-hydroxyvaline), can be used to synthesize α-hydroxy esterswhich have been found to induce increased skin thickness by increasingbiosyntheses of glycosaminoglycans, proteoglycans, collagen, elastin,and other dermal components. The compounds can be used to treat skindisorders such as age spots, skin lines, wrinkles, photoaging and aging.

[0007] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 is a schematic representation of the pantothenate andisoleucine-valine (ilv) biosynthetic pathways. Pantothenate biosyntheticenzymes are depicted in bold and their corresponding genes indicated initalics. Isoleucine-valine (ilv) biosynthetic enzymes are depicted inbold italics and their corresponding genes indicated in italics.

[0009]FIG. 2 is a schematic representation of the biosynthetic pathwayleading to [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid(“HMBPA”) in B. subtilis.

[0010]FIG. 3 is a schematic depiction of the structure of[R]-3-(2-hydroxy-3methyl-butyrylamino)-propionic acid (“HMBPA”).

[0011]FIG. 4 is a HPLC chromatogram of a sample of medium from a 14 Lfermentation of PA824.

[0012]FIG. 5 is a mass spectrum depicting the relative monoisotopic massof [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid.

[0013]FIG. 6 depicts an alignment of the C-terminal amino acids fromknown or suspected PanB proteins.

[0014]FIG. 7 is a schematic representation of the construction of theplasmid pAN624.

[0015]FIG. 8 is a schematic representation of the construction of theplasmid pAN620.

[0016]FIG. 9 is a schematic representation of the construction of theplasmid pAN636.

[0017]FIG. 10 is a schematic representation of the construction of theplasmid pAN637 which allows selection for single or multiple copiesusing chloramphenicol.

[0018]FIG. 11 is a schematic representation of the construction of theplasmid pAN238, a plasmid for overexpressing B. subtilis panE2 from theP₂₆ promoter.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is based, at least in part, on thediscovery of a novel biosynthetic pathway in bacteria, namely the[R]-3-(2-hydroxy-3-methyl-butyrylamino)propionic acid (“HMBPA”)biosynthetic pathway. In particular, it has been discovered thatbacteria are capable of generating HMBPA from α-ketoisovalerate (α-KIV),a key product of the isoleucine-valine (ilv) biosynthetic pathway andprecursor of the pantothenate biosynthetic pathway. Production of HMBPAin bacteria involves at least the pantothenate biosynthetic enzymesketopantoate reductase (the panE1 gene product) and/or acetohydroxyacidisomeroreductase (the ilvC gene product) and results from thecondensation of 2-hydroxyisovaleric acid (α-HIV), formed by reduction ofα-KIV, and β-alanine, the latter reaction being catalyzed by thepantothenate biosynthetic enzyme pantothenate synthetase (the panC geneproduct). Production of HMBPA is achieved by increasing ketopantoatereductase (e.g., PanE1) and/or PanE2 and/or acetohydroxyacidisomeroreductase activities (e.g., IlvC) in microorganisms, for example,by overexpressing or deregulating the genes encoding said enzymes.Optimal production of HMBPA is achieved by decreasing or deletingketopantoate hydroxymethyltransferase activity (the panB gene product)in microorganisms, for example, by modifying or deleting the panB genewhich encodes ketopantoate hydroxymethyltransferase (e.g., PanB),optionally in addition to increasing ketopantoate reductase and/or PanE2and/or acetohydroxyacid isomeroreductase activities in saidmicroorganisms. The substrates α-KIV and β-alanine are required forHMBPA production, the latter provided, for example, by β-alanine feedingand/or increased aspartate-α-decarboxylate activity (the panD geneproduct). Increasing substrate concentration (i.e., α-KIV and/orβ-alanine) further enhances production of HMBPA. α-KIV production can beincreased by overexpressing ilvBNCD genes and/or alsS. HMBPA productioncan further be increased by limiting serine availability or synthesis inappropriately engineered microorganisms.

[0020] In order that the present invention may be more readilyunderstood, certain terms are first defined herein.

[0021] The term “pantothenate biosynthetic pathway” includes thebiosynthetic pathway involving pantothenate biosynthetic enzymes (e.g.,polypeptides encoded by biosynthetic enzyme-encoding genes), compounds(e.g., precursors, substrates, intermediates or products), cofactors andthe like utilized in the formation or synthesis of pantothenate. Theterm “pantothenate biosynthetic pathway” includes the biosyntheticpathway leading to the synthesis of pantothenate in microorganisms(e.g., in vivo) as well as the biosynthetic pathway leading to thesynthesis of pantothenate in vitro.

[0022] The term “pantothenate biosynthetic enzyme” includes any enzymeutilized in the formation of a compound (e.g., intermediate or product)of the pantothenate biosynthetic pathway. For example, synthesis ofpantoate from α-ketoisovalerate (α-KIV) proceeds via the intermediate,ketopantoate. Formation of ketopantoate is catalyzed by the pantothenatebiosynthetic enzyme ketopantoate hydroxymethyltransferase (the panB geneproduct). Formation of pantoate is catalyzed by the pantothenatebiosynthetic enzyme ketopantoate reductase (the panE gene product).Synthesis of β-alanine from aspartate is catalyzed by the pantothenatebiosynthetic enzyme aspartate-α-decarboxylase (the panD gene product).Formation of pantothenate from pantoate and β-alanine (e.g.,condensation) is catalyzed by the pantothenate biosynthetic enzymepantothenate synthetase (the panC gene product). Based on the newlydiscovered HMBPA biosynthesis pathway, pantothenate biosynthetic enzymesmay also perform an alternative function as enzymes in the HMBPAbiosynthetic pathway described herein.

[0023] The term “pantothenate” includes the free acid form ofpantothenate, also referred to as “pantothenic acid” as well as any saltthereof (e.g., derived by replacing the acidic hydrogen of pantothenateor pantothenic acid with a cation, for example, calcium, sodium,potassium, ammonium), also referred to as a “pantothenate salt”. Theterm “pantothenate” also includes alcohol derivatives of pantothenate.Preferred pantothenate salts are calcium pantothenate or sodiumpantothenate. A preferred alcohol derivative is pantothenol.Pantothenate salts and/or alcohols of the present invention includesalts and/or alcohols prepared via conventional methods from the freeacids described herein. In another embodiment, calcium pantothenate issynthesized directly by a microorganism of the present invention. Apantothenate salt of the present invention can likewise be converted toa free acid form of pantothenate or pantothenic acid by conventionalmethodology.

[0024] The term “isoleucine-valine biosynthetic pathway” includes thebiosynthetic pathway involving isoleucine-valine biosynthetic enzymes(e.g., polypeptides encoded by biosynthetic enzyme-encoding genes),compounds (e.g., precursors, substrates, intermediates or products),cofactors and the like utilized in the formation or synthesis ofconversion of pyruvate to valine or isoleucine. The term“isoleucine-valine biosynthetic pathway” includes the biosyntheticpathway leading to the synthesis of valine or isoleucine inmicroorganisms (e.g., in vivo) as well as the biosynthetic pathwayleading to the synthesis of valine or isoleucine in vitro. FIG. 1includes a schematic representation of the isoleucine-valinebiosynthetic pathway. Isoleucine-valine biosynthetic enzymes aredepicted in bold italics and their corresponding genes indicated initalics

[0025] The term “isoleucine-valine biosynthetic enzyme” includes anyenzyme utilized in the formation of a compound (e.g., intermediate orproduct) of the isoleucine-valine biosynthetic pathway. According toFIG. 1, synthesis of valine from pyruvate proceeds via theintermediates, acetolactate, α,β-dihydroxyisovalerate (α,β-DHIV) andα-ketoisovalerate (α-KIV). Formation of acetolactate from pyruvate iscatalyzed by the isoleucine-valine biosynthetic enzyme acetohydroxyacidsynthetase (the ilvBN gene product, or alternatively, the alsS geneproduct). Formation of α,β-DHIV from acetolactate is catalyzed by theisoleucine-valine biosynthetic enzyme acetohydroxyacidisomero reductase(the ilvC gene product). Synthesis of α-KIV from α,β-DHIV is catalyzedby the isoleucine-valine biosynthetic enzyme dihydroxyacid dehydratase(the ilvD gene product). Moreover, valine and isoleucine can beinterconverted with their respective α-keto compounds by branched chainamino acid transaminases. Based on the newly discovered HMBPAbiosynthesis pathway, isoleucine-valine biosynthetic enzymes may alsoperform an alternative function as enzymes in the HMBPA biosyntheticpathway described herein.

[0026] The term “3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid(“HMBPA”) biosynthetic pathway” includes the alternative biosyntheticpathway involving biosynthetic enzymes and compounds (e.g., substratesand the like) traditionally associated with the pantothenatebiosynthetic pathway utilized in the formation or synthesis of HMBPA.The term “HMBPA biosynthetic pathway” includes the biosynthetic pathwayleading to the synthesis of HMBPA in microorganisms (e.g., in vivo) aswell as the biosynthetic pathway leading to the synthesis of HMBPA invitro.

[0027] The term “HMBPA biosynthetic enzyme” includes any enzyme utilizedin the formation of a compound (e.g., intermediate or product) of theHMBPA biosynthetic pathway. For example, synthesis of2-hydroxyisovaleric acid (α-HIV) from α-ketoisovalerate (α-KIV) iscatalyzed by the panE1 or panE2 gene product (PanE1, alternativelyreferred to herein ketopantoate reductase or PanE2, a α-ketoacidreductase that does not significantly contribute to pantothenatebiosynthesis) and/or is catalyzed by the ilvC gene product(alternatively referred to herein as acetohydroxyacid isomeroreductase).Formation of HMBPA from β-alanine and α-HIV is catalyzed by the panCgene product (alternatively referred to herein as pantothenatesynthetase).

[0028] The term “3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid(“HMBPA”)” includes the free acid form of HMBPA, also referred to as“3-(2-hydroxy-3-methyl-butyrylamino)-propionate” as well as any saltthereof (e.g., derived by replacing the acidic hydrogen of[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid or[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionate with a cation, forexample, calcium, sodium, potassium, ammonium), also referred to as a“3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid salt” or “HMBPAsalt”. Preferred HMBPA salts are calcium HMBPA or sodium HMBPA. HMBPAsalts of the present invention include salts prepared via conventionalmethods from the free acids described herein. An HMBPA salt of thepresent invention can likewise be converted to a free acid form of[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid or[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionate by conventionalmethodology.

[0029] Various aspects of the invention are described in further detailin the following subsections.

[0030] I. Targeting Genes Encoding Various Pantothenate and/orIsoleucine-Valine(ilv) and/or HMBPA Biosynthetic Enzymes

[0031] In one embodiment, the present invention features targeting ormodifying various biosynthetic enzymes of the pantothenate and/orisoleucine-valine(ilv) and/or HMBPA biosynthetic pathways. Inparticular, the invention features modifying various enzymaticactivities associated with said pathways by modifying or altering thegenes encoding said biosynthetic enzymes.

[0032] The term “gene”, as used herein, includes a nucleic acid molecule(e.g., a DNA molecule or segment thereof) that, in an organism, can beseparated from another gene or other genes, by intergenic DNA (i.e.,intervening or spacer DNA which naturally flanks the gene and/orseparates genes in the chromosomal DNA of the organism). Alternatively,a gene may slightly overlap another gene (e.g., the 3′ end of a firstgene overlapping the 5′ end of a second gene), said overlapping genesseparated from other genes by intergenic DNA. A gene may directsynthesis of an enzyme or other protein molecule (e.g., may comprisecoding seqeunces, for example, a contiguous open reading frame (ORF)which encodes a protein) or may itself be functional in the organism. Agene in an organism, may be clustered in an operon, as defined herein,said operon being separated from other genes and/or operons by theintergenic DNA. An “isolated gene”, as used herein, includes a genewhich is essentially free of sequences which naturally flank the gene inthe chromosomal DNA of the organism from which the gene is derived(i.e., is free of adjacent coding sequences which encode a second ordistinct protein, adjacent structural sequences or the like) andoptionally includes 5′ and 3′ regulatory sequences, for example promotersequences and/or terminator sequences. In one embodiment, an isolatedgene includes predominantly coding sequences for a protein (e.g.,sequences which encode Bacillus proteins). In another embodiment, anisolated gene includes coding sequences for a protein (e.g., for aBacillus protein) and adjacent 5′ and/or 3′ regulatory sequences fromthe chromosomal DNA of the organism from which the gene is derived(e.g., adjacent 5′ and/or 3′ Bacillus regulatory sequences). Preferably,an isolated gene contains less than about 10 kb, 5 kb, 2 kb, 1 kb, 0.5kb, 0.2 kb, 0.1 kb, 50 bp, 25 bp or 10 bp of nucleotide sequences thatnaturally flank the gene in the chromosomal DNA of the organism fromwhich the gene is derived.

[0033] The term “operon” includes at least two adjacent genes or ORFs,optionally overlapping in sequence at either the 5′ or 3′ end of atleast one gene or ORF. The term “operon” includes a coordinated unit ofgene expression that contains a promoter and possibly a regulatoryelement associated with one or more adjacent genes or ORFs (e.g.,structural genes encoding enzymes, for example, biosynthetic enzymes).Expression of the genes (e.g., structural genes) can be coordinatelyregulated, for example, by regulatory proteins binding to the regulatoryelement or by anti-termination of transcription. The genes of an operon(e.g., structural genes) can be transcribed to give a single mRNA thatencodes all of the proteins.

[0034] A “gene having a mutation” or “mutant gene” as used herein,includes a gene having a nucleotide sequence which includes at least onealteration (e.g., substitution, insertion, deletion) such that thepolypeptide or protein encoded by said mutant exhibits an activity thatdiffers from the polypeptide or protein encoded by the wild-type nucleicacid molecule or gene. In one embodiment, a gene having a mutation ormutant gene encodes a polypeptide or protein having an increasedactivity as compared to the polypeptide or protein encoded by thewild-type gene, for example, when assayed under similar conditions(e.g., assayed in microorganisms cultured at the same temperature). Asused herein, an “increased activity” or “increased enzymatic activity”is one that is at least 5% greater than that of the polypeptide orprotein encoded by the wild-type nucleic acid molecule or gene,preferably at least 5-10% greater, more preferably at least 10-25%greater and even more preferably at least 25-50%, 50-75% or 75-100%greater than that of the polypeptide or protein encoded by the wild-typenucleic acid molecule or gene. Ranges intermediate to the above-recitedvalues, e.g., 75-85%, 85-90%, 90-95%, are also intended to beencompassed by the present invention. As used herein, an “increasedactivity” or “increased enzymatic activity” can also include an activitythat is at least 1.25-fold greater than the activity of the polypeptideor protein encoded by the wild-type gene, preferably at least 1.5-foldgreater, more preferably at least 2-fold greater and even morepreferably at least 3-fold, 4-fold, 5-fold, 10-fold, 20fold, 50-fold,100-fold or greater than the activity of the polypeptide or proteinencoded by the wild-type gene.

[0035] In another embodiment, a gene having a mutation or mutant geneencodes a polypeptide or protein having a reduced activity as comparedto the polypeptide or protein encoded by the wild-type gene, forexample, when assayed under similar conditions (e.g., assayed inmicroorganisms cultured at the same temperature). A mutant gene also canencode no polypeptide or have a reduced level of production of thewild-type polypeptide. As used herein, a “reduced activity” or “reducedenzymatic activity” is one that is at least 5% less than that of thepolypeptide or protein encoded by the wild-type nucleic acid molecule orgene, preferably at least 5-10% less, more preferably at least 10-25%less and even more preferably at least 25-50%, 50-75% or 75-100% lessthan that of the polypeptide or protein encoded by the wild-type nucleicacid molecule or gene. Ranges intermediate to the above-recited values,e.g., 75-85%, 85-90%, 90-95%, are also intended to be encompassed by thepresent invention. As used herein, a “reduced activity” or “reducedenzymatic activity” can also include an activity that has been deletedor “knocked out” (e.g., approximately 100% less activity than that ofthe polypeptide or protein encoded by the wild-type nucleic acidmolecule or gene).

[0036] Activity can be determined according to any well accepted assayfor measuring activity of a particular protein of interest. Activity canbe measured or assayed directly, for example, measuring an activity of aprotein isolated or purified from a cell or mocroorganism.Alternatively, an activity can be measured or assayed within a cell ormocroorganism or in an extracellular medium. For example, assaying for amutant gene (i.e., said mutant encoding a reduced enzymatic activity)can be accomplished by expressing the mutated gene in a microorganism,for example, a mutant microorganism in which the enzyme istemperature-sensitive, and assaying the mutant gene for the ability tocomplement a temperature sensitive (Ts) mutant for enzymatic activity. Amutant gene that encodes an “increased enzymatic activity” can be onethat complements the Ts mutant more effectively than, for example, acorresponding wild-type gene. A mutant gene that encodes a “reducedenzymatic activity” is one that complements the Ts mutant lesseffectively than, for example, a corresponding wild-type gene.

[0037] It will be appreciated by the skilled artisan that even a singlesubstitution in a nucleic acid or gene sequence (e.g., a basesubstitution that encodes an amino acid change in the correspondingamino acid sequence) can dramatically affect the activity of an encodedpolypeptide or protein as compared to the corresponding wild-typepolypeptide or protein. A mutant gene (e.g., encoding a mutantpolypeptide or protein), as defined herein, is readily distinguishablefrom a nucleic acid or gene encoding a protein homologue in that amutant gene encodes a protein or polypeptide having an altered activity,optionally observable as a different or distinct phenotype in amicroorganism expressing said mutant gene or producing said mutantprotein or polypeptide (i.e., a mutant microorganism) as compared to acorresponding microorganism expressing the wild-type gene. By contrast,a protein homologue has an identical or substantially similar activity,optionally phenotypically indiscernable when produced in amicroorganism, as compared to a corresponding microorganism expressingthe wild-type gene. Accordingly it is not, for example, the degree ofsequence identity between nucleic acid molecules, genes, protein orpolypeptides that serves to distinguish between homologues and mutants,rather it is the activity of the encoded protein or polypeptide thatdistinguishes between homologues and mutants: homologues having, forexample, low (e.g., 30-50% sequence identity) sequence identity yethaving substantially equivalent functional activities, and mutants, forexample sharing 99% sequence identity yet having dramatically differentor altered functional activities.

[0038] It will also be appreciated by the skilled artisan that nucleicacid molecules, genes, protein or polypeptides for use in the instantinvention can be derived from any microorganisms having a HMBPAbiosynthetic pathway, an ilv biosynthetic pathway or a pantothenatebiosynthetic pathway. Such nucleic acid molecules, genes, protein orpolypeptides can be identified by the skilled artisan using Blowntechniques such as homology screening, sequence comparison and the like,and can be modified by the skilled artisan in such a way that expressionor production of these nucleic acid molecules, genes, protein orpolypeptides occurs in a recombinant microorganism (e.g., by usingappropriate promoters, ribosomal binding sites, expression orintegration vectors, modifying the sequence of the genes such that thetranscription is increased (taking into account the preferable codonusage), etc., according to techniques described herein and those knownin the art).

[0039] In one embodiment, the genes of the present invention are derivedfrom a Gram positive microorganism organism (e.g., a microorganism whichretains basic dye, for example, crystal violet, due to the presence of aGram-positive wall surrounding the microorganism). The term “derivedfrom” (e.g., “derived from” a Gram positive microorganism) refers to agene which is naturally found in the microorganism (e.g., is naturallyfound in a Gram positive microorganism). In a preferred embodiment, thegenes of the present invention are derived from a microorganismbelonging to a genus selected from the group consisting of Bacillus,Cornyebacterium (e.g., Cornyebacterium glutamicum), Lactobacillus,Lactococci and Streptomyces. In a more preferred embodiment, the genesof the present invention are derived from a microorganism is of thegenus Bacillus. In another preferred embodiment, the genes of thepresent invention are derived from a microorganism selected from thegroup consisting of Bacillus subtilis, Bacillus lentimorbus, Bacilluslentus, Bacillus firmus, Bacillus pantothenticus, Bacillusamyloliquefaciens, Bacillus cereus, Bacillus circulans, Bacilluscoagulans, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus thuringiensis, Bacillus halodurans, and other Group 1Bacillus species, for example, as characterized by 16S rRNA type. Inanother preferred embodiment, the gene is derived from Bacillus brevisor Bacillus stearothermophilus. In another preferred embodiment, thegenes of the present invention are derived from a microorganism selectedfrom the group consisting of Bacillus licheniformis, Bacillusamyloliquefaciens, Bacillus subtilis, and Bacillus pumilus. In aparticularly preferred embodiment, the gene is derived from Bacillussubtilis (e.g., is Bacillus subtilis-derived). The term “derived fromBacillus subtilis” or “Bacillus's subtilis-derived” includes a genewhich is naturally found in the microorganism Bacillus subtilis.Included within the scope of the present invention are Bacillus-derivedgenes (e.g., B. subtilis-derived genes), for example, Bacillus or B.subtilis coaX genes, serA genes, glyA genes, coaA genes, pan genesand/or ilv genes.

[0040] In another embodiment, the genes of the present invention arederived from a Gram negative (excludes basic dye) microorganism. In apreferred embodiment, the genes of the present invention are derivedfrom a microorganism belonging to a genus selected from the groupconsisting of Salmonella (e.g., Salmonella typhimurium), Escherichia,Klebsiella, Serratia, and Proteus. In a more preferred embodiment, thegenes of the present invention are derived from a microorganism of thegenus Escherichia. In an even more preferred embodiment, the genes ofthe present invention are derived from Escherichia coli. In anotherembodiment, the genes of the present invention are derived fromSaccharomyces (e.g., Saccharomyces cerevisiae).

[0041] II. Recombinant Nucleic Acid Molecules and Vectors

[0042] The present invention further features recombinant nucleic acidmolecules (e.g., recombinant DNA molecules) that include genes describedherein (e.g., isolated genes), preferably Bacillus genes, morepreferably Bacillus subtilis genes, even more preferably Bacillussubtilis pantothenate biosynthetic genes and/or isoleucine-valine (ilv)biosynthetic genes and/or HMBPA biosynthetic genes. The term“recombinant nucleic acid molecule” includes a nucleic acid molecule(e.g., a DNA molecule) that has been altered, modified or engineeredsuch that it differs in nucleotide sequence from the native or naturalnucleic acid molecule from which the recombinant nucleic acid moleculewas derived (e.g., by addition, deletion or substitution of one or morenucleotides). Preferably, a recombinant nucleic acid molecule (e.g., arecombinant DNA molecule) includes an isolated gene of the presentinvention operably linked to regulatory sequences. The phrase “operablyliked to regulatory sequence(s)” means that the nucleotide sequence ofthe gene of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression (e.g., enhanced, increased, constitutive,basal, attenuated, decreased or repressed expression) of the gene,preferably expression of a gene product encoded by the gene (e.g., whenthe recombinant nucleic acid molecule is included in a recombinantvector, as defined herein, and is introduced into a microorganism).

[0043] The term “regulatory sequence” includes nucleic acid sequenceswhich affect (e.g., modulate or regulate) expression of other nucleicacid sequences (i.e., genes). In one embodiment, a regulatory sequenceis included in a recombinant nucleic acid molecule in a similar oridentical position and/or orientation relative to a particular gene ofinterest as is observed for the regulatory sequence and gene of interestas it appears in nature, e.g., in a native position and/or orientation.For example, a gene of interest can be included in a recombinant nucleicacid molecule operably linked to a regulatory sequence which accompaniesor is adjacent to the gene of interest in the natural organism (e.g.,operably linked to “native” regulatory sequences (e.g., to the “native”promoter). Alternatively, a gene of interest can be included in arecombinant nucleic acid molecule operably linked to a regulatorysequence which accompanies or is adjacent to another (e.g., a different)gene in the natural organism. Alternatively, a gene of interest can beincluded in a recombinant nucleic acid molecule operably linked to aregulatory sequence from another organism. For example, regulatorysequences from other microbes (e.g., other bacterial regulatorysequences, bacteriophage regulatory sequences and the like) can beoperably linked to a particular gene of interest.

[0044] In one embodiment, a regulatory sequence is a non-native ornon-naturally-occurring sequence (e.g., a sequence which has beenmodified, mutated, substituted, derivatized, deleted including sequenceswhich are chemically synthesized). Preferred regulatory sequencesinclude promoters, enhancers, termination signals, anti-terminationsignals and other expression control elements (e.g., sequences to whichrepressors or inducers bind and/or binding sites for transcriptionaland/or translational regulatory proteins, for example, in thetranscribed mRNA). Such regulatory sequences are described, for example,in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Regulatorysequences include those which direct constitutive expression of anucleotide sequence in a microorganism (e.g., constitutive promoters andstrong constitutive promoters), those which direct inducible expressionof a nucleotide sequence in a microorganism (e.g., inducible promoters,for example, xylose inducible promoters) and those which attenuate orrepress expression of a nucleotide sequence in a microorganism (e.g.,attenuation signals or repressor sequences). It is also within the scopeof the present invention to regulate expression of a gene of interest byremoving or deleting regulatory sequences. For example, sequencesinvolved in the negative regulation of transcription can be removed suchthat expression of a gene of interest is enhanced.

[0045] In one embodiment, a recombinant nucleic acid molecule of thepresent invention includes a nucleic acid sequence or gene that encodeat least one bacterial gene product (e.g., a pantothenate biosyntheticenzyme, an isoleucine-valine biosynthetic enzyme and/or a HMBPAbiosynthetic enzyme) operably linked to a promoter or promoter sequence.Preferred promoters of the present invention include Bacillus promotersand/or bacteriophage promoters (e.g., bacteriophage which infectBacillus). In one embodiment, a promoter is a Bacillus promoter,preferably a strong Bacillus promoter (e.g., a promoter associated witha biochemical housekeeping gene in Bacillus or a promoter associatedwith a glycolytic pathway gene in Bacillus). In another embodiment, apromoter is a bacteriophage promoter. In a preferred embodiment, thepromoter is from the bacteriophage SP01. In a particularly preferredembodiment, a promoter is selected from the group consisting of P₁₅, P₂₆or P_(veg), having for example, the following respective seqeunces:GCTATTGACGACAGCTATGGTTCACTGTCCACCAACCAAAACTGTGCTCAGTACCGCCAATATTTCTCCCTTGAGGGGTACAAAGAGGTGTCCCTAGAAGAGATCCACGCTGTGTAAAAATTTTACAAAAAGGTATTGACTTTCCCTACAGGGTGTGTAATAATTTAATTACAGGCGGGGGCAACCCCGCCTGT(SEQ ID NO:1),GCCTACCTAGCTTCCAAGAAAGATATCCTAACAGCACAAGAGCGGAAAGATGTTTTGTTCTACATCCAGAACAACCTCTGCTAAAATTCCTGAAAAATTTTGCAAAAAGTTGTTGACTTTATCTACAAGGTGTGGTATAATAATCTTAACAACAGC AGGACGC (SEQ IDNO:2), and GAGGAATCATAGAATTTTGTCAAAATAATTTTATTGACAACGTCTTATTAACGTTGATATAATTTAAATTTTATTTGACAAAAATGGGCTCGTGTTGTACAATAAATGTAGTGAGGTGGATGCAATG (SEQ ID NO:3). Additional preferred promotersinclude tef (the translational elongation factor (TEF) promoter) and pyc(the pyruvate carboxylase (PYC) promoter), which promote high levelexpression in Bacillus (e.g., Bacillus subtilis). Additional preferredpromoters, for example, for use in Gram positive microorganisms include,but are not limited to, amy and SPO2 promoters. Additional preferredpromoters, for example, for use in Gram negative microorganisms include,but are not limited to, cos, tac, trp, tei, trp-tet, lpp, lac, lpp-lac,lacIQ, T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL.

[0046] In another embodiment, a recombinant nucleic acid molecule of thepresent invention includes a terminator sequence or terminator sequences(e.g., transcription terminator sequences). The term “terminatorsequences” includes regulatory sequences that serve to terminatetranscription of mRNA. Terminator sequences (or tandem transcriptionterminators) can further serve to stabilize mRNA (e.g., by addingstructure to mRNA), for example, against nucleases.

[0047] In yet another embodiment, a recombinant nucleic acid molecule ofthe present invention includes sequences which allow for detection ofthe vector containing said sequences (i.e., detectable and/or selectablemarkers), for example, genes that encode antibiotic resistance orsequences that overcome auxotrophic mutations, for example, trpC,fluorescent markers, drug markers, and/or calorimetric markers (e.g.,lacZ/β-galactosidase). In yet another embodiment, a recombinant nucleicacid molecule of the present invention includes an artificial ribosomebinding site (RBS) or a sequence that becomes transcribed into anartificial RBS. The term “artificial ribosome binding site (RBS)”includes a site within an mRNA molecule (e.g., coded within DNA) towhich a ribosome binds (e.g., to initiate translation) which differsfrom a native RBS (e.g., a RBS found in a naturally-occurring gene) byat least one nucleotide. Preferred artificial RBSs include about 5-6,7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26,27-28, 29-30 or more nucleotides of which about 1-2, 3-4, 5-6, 7-8,9-10, 11-12, 13-15 or more differ from the native RBS (e.g., the nativeRBS of a gene of interest, for example, the native panB RBSTAAACATGAGGAGGAGAAAACATG (SEQ ID NO:4) or the native panD RBSATTCGAGAAATGGAGAGAATATAATATG (SEQ ID NO:5)).

[0048] Preferably, nucleotides that differ are substituted such thatthey are identical to one or more nucleotides of an ideal RBS whenoptimally aligned for comparisons. Ideal RBSs include, but are notlimited to, AGAAAGGAGGTGA (SEQ ID NO:6), TTAAGAAAGGAGGTGANNNNATG (SEQ IDNO:7), TTAGAAAGGAGGTGANNNNNATG (SEQ ID NO:8), AGAAAGGAGGTGANNNNNNNATG(SEQ ID NO:9), and AGAAAGGAGGTGANNNNNNATG (SEQ ID NO:10). ArtificialRBSs can be used to replace the naturally-occurring or native RBSsassociated with a particular gene. Artificial RBSs preferably increasetranslation of a particular gene. Preferred artificial RBSs (e.g., RBSsfor increasing the translation of panB, for example, of B. subtilispanB) include CCCTCTAGAAGGAGGAGAAAACATG (SEQ ID NO:11) andCCCTCTAGAGGAGGAGAAAACATG (SEQ ID NO:12). Preferred artificial RBSs(e.g., RBSs for increasing the translation of panD, for example, of B.subtilis panD) include TTAGAAAGGAGGATTTAAATATG (SEQ ID NO:13),TTAGAAAGGAGGTTTAATTAATG (SEQ ID NO:14), TTAGAAAGGAGGTGATTTAAATG (SEQ IDNO:15), TTAGAAAGGAGGTGTTTAAAATG (SEQ ID NO:16), ATTCGAGAAAGGAGGTGAATATAATATG (SEQ ID NO:17), ATTCGAGAAAGGAGGTGAATAATAATG (SEQ IDNO:18), and ATTCGTAGAAAGGAGGTGAATTAATATG (SEQ ID NO:19).

[0049] The present invention further features vectors (e.g., recombinantvectors) that include nucleic acid molecules (e.g., genes or recombinantnucleic acid molecules comprising said genes) as described herein. Theterm “recombinant vector” includes a vector (e.g., plasmid, phage,phasmid, virus, cosmid or other purified nucleic acid vector) that hasbeen altered, modified or engineered such that it contains greater,fewer or different nucleic acid sequences than those included in thenative or natural nucleic acid molecule from which the recombinantvector was derived. Preferably, the recombinant vector includes abiosynythetic enzyme-encoding gene or recombinant nucleic acid moleculeincluding said gene, operably linked to regulatory sequences, forexample, promoter sequences, terminator sequences and/or artificialribosome binding sites (RBSs), as defined herein. In another embodiment,a recombinant vector of the present invention includes sequences thatenhance replication in bacteria (e.g., replication-enhancing sequences).In one embodiment, replication-enhancing sequences are derived from E.coli. In another embodiment, replication-enhancing sequences are derivedfrom pBR322.

[0050] In yet another embodiment, a recombinant vector of the presentinvention includes antibiotic resistance sequences. The term “antibioticresistance sequences” includes sequences which promote or conferresistance to antibiotics on the host organism (e.g., Bacillus). In oneembodiment, the antibiotic resistance sequences are selected from thegroup consisting of cat (chloramphenicol resistance) sequences, tet(tetracycline resistance) sequences, erm (erythromycin resistance)sequences, neo (neomycin resistance) sequences, kan (kanamycinresistance) and spec (spectinomycin resistance) sequences. Recombinantvectors of the present invention can further include homologousrecombination sequences (e.g., sequences designed to allow recombinationof the gene of interest into the chromosome of the host organism). Forexample, bpr, vpr, and/or amyE sequences can be used as homology targetsfor recombination into the host chromosome. It will further beappreciated by one of skill in the art that the design of a vector canbe tailored depending on such factors as the choice of microorganism tobe genetically engineered, the level of expression of gene productdesired and the like.

[0051] IV. Recombinant Microorganisms

[0052] The present invention further features microorganisms, i.e.,recombinant microorganisms, that include vectors or genes (e.g.,wild-type and/or mutated genes) as described herein. As used herein, theterm “recombinant microorganism” includes a microorganism (e.g.,bacteria, yeast cell, fungal cell, etc.) that has been geneticallyaltered, modified or engineered (e.g., genetically engineered) such thatit exhibits an altered, modified or different genotype and/or phenotype(e.g., when the genetic modification affects coding nucleic acidsequences of the microorganism) as compared to the naturally-occurringmicroorganism from which it was derived.

[0053] In one embodiment, a recombinant microorganism of the presentinvention is a Gram positive organism (e.g., a microorganism whichretains basic dye, for example, crystal violet, due to the presence of aGram-positive wall surrounding the microorganism). In a preferredembodiment, the recombinant microorganism is a microorganism belongingto a genus selected from the group consisting of Bacillus,Cornyebacterium, Lactobacillus, Lactococci and Streptomyces. In a morepreferred embodiment, the recombinant microorganism is of the genusBacillus. In another preferred embodiment, the recombinant microorganismis selected from the group consisting of Bacillus subtilis, Bacilluslentimorbus, Bacillus lentus, Bacillus firmus, Bacillus pantothenticus,Bacillus amyloliquefaciens, Bacillus cereus, Bacillus circulans,Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium,Bacillus pumilus, Bacillus thuringiensis, Bacillus halodurans, and otherGroup 1 Bacillus species, for example, as characterized by 16S rRNAtype. In another preferred embodiment, the recombinant microorganism isBacillus brevis or Bacillus stearothermophilis. In another preferredembodiment, the recombinant microorganism is selected from the groupconsisting of Bacillus licheniformis, Bacillus amyloliquefaciens,Bacillus subtilis, and Bacillus pumilus.

[0054] In another embodiment, the recombinant microorganism is a Gramnegative (excludes basic dye) organism. In a preferred embodiment, therecombinant microorganism is a microorganism belonging to a genusselected from the group consisting of Salmonella, Escherichia,Klebsiella, Serratia, and Proteus. In a more preferred embodiment, therecombinant microorganism is of the genus Escherichia. In an even morepreferred embodiment, the recombinant microorganism is Escherichia coli.In another embodiment, the recombinant microorganism is Saccharomyces(e.g., S. cerevisiae).

[0055] A preferred “recombinant” microorganism of the present inventionis a microorganism having a deregulated pantothenate biosynthesispathway or enzyme, a deregulated isoleucine-valine (ilv) biosyntheticpathway or enzyme and/or a deregulated HMBPA biosynthetic pathway orenzyme. The term “deregulated” or “deregulation” includes the alterationor modification of at least one gene in a microorganism that encodes anenzyme in a biosynthetic pathway, such that the level or activity of thebiosynthetic enzyme in the microorganism is altered or modified.Preferably, at least one gene that encodes an enzyme in a biosyntheticpathway is altered or modified such that the gene product is enhanced orincreased. The phrase “deregulated pathway” can also include abiosynthetic pathway in which more than one gene that encodes an enzymein a biosynthetic pathway is altered or modified such that the level oractivity of more than one biosynthetic enzyme is altered or modified.The ability to “deregulate” a pathway (e.g., to simultaneouslyderegulate more than one gene in a given biosynthetic pathway) in amicroorganism in some cases arises from the particular phenomenon ofmicroorganisms in which more than one enzyme (e.g., two or threebiosynthetic enzymes) are encoded by genes occurring adjacent to oneanother on a contiguous piece of genetic material termed an “operon”(defined herein). Due to the coordinated regulation of genes included inan operon, alteration or modification of the single promoter and/orregulatory element can result in alteration or modification of theexpression of each gene product encoded by the operon. Alteration ormodification of the regulatory element can include, but is not limitedto removing the endogenous promoter and/or regulatory element(s), addingstrong promoters, inducible promoters or multiple promoters or removingregulatory sequences such that expression of the gene products ismodified, modifying the chromosomal location of the operon, alteringnucleic acid sequences adjacent to the operon or within the operon suchas a ribosome binding site, increasing the copy number of the operon,modifying proteins (e.g., regulatory proteins, suppressors, enhancers,transcriptional activators and the like) involved in transcription ofthe operon and/or translation of the gene products of the operon, or anyother conventional means of deregulating expression of genes routine inthe art (including but not limited to use of antisense nucleic acidmolecules, for example, to block expression of repressor proteins).Deregulation can also involve altering the coding region of one or moregenes to yield, for example, an enzyme that is feedback resistant or hasa higher or lower specific activity.

[0056] In another preferred embodiment, a recombinant microorganism isdesigned or engineered such that at least one pantothenate biosyntheticenzyme, at least one isoleucine-valine biosynthetic enzyme, and/or atleast one HMBPA biosynthetic enzyme is overexpressed. The term“overexpressed” or “overexpression” includes expression of a geneproduct (e.g., a biosynthetic enzyme) at a level greater than thatexpressed prior to manipulation of the microorganism or in a comparablemicroorganism which has not been manipulated. In one embodiment, themicroorganism can be genetically designed or engineered to overexpress alevel of gene product greater than that expressed in a comparablemicroorganism which has not been engineered.

[0057] Genetic engineering can include, but is not limited to, alteringor modifying regulatory sequences or sites associated with expression ofa particular gene (e.g., by adding strong promoters, inducible promotersor multiple promoters or by removing regulatory sequences such thatexpression is constitutive), modifying the chromosomal location of aparticular gene, altering nucleic acid sequences adjacent to aparticular gene such as a ribosome binding site, increasing the copynumber of a particular gene, modifying proteins (e.g., regulatoryproteins, suppressors, enhancers, transcriptional activators and thelike) involved in transcription of a particular gene and/or translationof a particular gene product, or any other conventional means ofderegulating expression of a particular gene routine in the art(including but not limited to use of antisense nucleic acid molecules,for example, to block expression of repressor proteins). Geneticengineering can also include deletion of a gene, for example, to block apathway or to remove a repressor. In embodiments featuringmicroorganisms having deleted genes, the skilled artisan will appreciatethat at least low levels of certain compounds may be required to bepresent in or added to the culture medium in order that the viability ofthe microorganism is not compromised. Often, such low levels are presentin complex culture media as routinely formulated. Moreover, in processesfeaturing culturing microorganisms having deleted genes cultured underconditions such that commercially or industrially attractive quantitiesof product are produced, it may be necessary to supplement culture mediawith slightly increased levels of certain compounds. For example, inprocesses featuring culturing a microorganism having a deleted panBgene, at least low levels of pantothenate must be present in the media,e.g., levels such as those found in routinely formulated complex media,whereas slightly increased levels of pantothenate may be added to themedia in order to produce commercially or industrially attractiveamounts of, for example, HMBPA. For example, 10-30 mg/L pantothenate canbe added to the media in order to produce commercially or industriallyattractive amounts of HMBPA.

[0058] In another embodiment, the microorganism can be physically orenvironmentally manipulated to overexpress a level of gene productgreater than that expressed prior to manipulation of the microorganismor in a comparable microorganism which has not been manipulated. Forexample, a microorganism can be treated with or cultured in the presenceof an agent known or suspected to increase transcription of a particulargene and/or translation of a particular gene product such thattranscription and/or translation are enhanced or increased.Alternatively, a microorganism can be cultured at a temperature selectedto increase transcription of a particular gene and/or translation of aparticular gene product such that transcription and/or translation areenhanced or increased.

[0059] V. Culturing and Fermenting Recombinant Microorganisms

[0060] The term “culturing” includes maintaining and/or growing a livingmicroorganism of the present invention (e.g., maintaining and/or growinga culture or strain). In one embodiment, a microorganism of theinvention is cultured in liquid media. In another embodiment, amicroorganism of the invention is cultured in solid media or semi-solidmedia. In a preferred embodiment, a microorganism of the invention iscultured in media (e.g., a sterile, liquid media) comprising nutrientsessential or beneficial to the maintenance and/or growth of themicroorganism (e.g., carbon sources or carbon substrate, for examplecarbohydrate, hydrocarbons, oils, fats, fatty acids, organic acids, andalcohols; nitrogen sources, for example, peptone, yeast extracts, meatextracts, malt extracts, urea, ammonium sulfate, ammonium chloride,ammonium nitrate and ammonium phosphate; phosphorus sources, forexample, phosphoric acid, sodium and potassium salts thereof, traceelements, for example, magnesium, iron, manganese, calcium, copper,zinc, boron, molybdenum, and/or cobalt salts; as well as growth factorssuch as amino acids, vitamins, growth promoters and the like).

[0061] Preferably, microorganisms of the present invention are culturedunder controlled pH. The term “controlled pH” includes any pH whichresults in production of the desired product (e.g., HMBPA). In oneembodiment microorganisms are cultured at a pH of about 7. In anotherembodiment, microorganisms are cultured at a pH of between 6.0 and 8.5.The desired pH may be maintained by any number of methods known to thoseskilled in the art.

[0062] Also preferably, microorganisms of the present invention arecultured under controlled aeration. The term “controlled aeration”includes sufficient aeration (e.g., oxygen) to result in production ofthe desired product (e.g., HMBPA). In one embodiment, aeration iscontrolled by regulating oxygen levels in the culture, for example, byregulating the amount of oxygen dissolved in culture media. Preferably,aeration of the culture is controlled by agitating the culture.Agitation may be provided by a propeller or similar mechanical agitationequipment, by revolving or shaking the cuture vessel (e.g., tube orflask) or by various pumping equipment. Aeration may be furthercontrolled by the passage of sterile air or oxygen through the medium(e.g., through the fermentation mixture). Also preferably,microorganisms of the present invention are cultured without excessfoaming (e.g., via addition of antifoaming agents).

[0063] Moreover, microorganisms of the present invention can be culturedunder controlled temperatures. The term “controlled temperature”includes any temperature which results in production of the desiredproduct (e.g., HMBPA). In one embodiment, controlled temperaturesinclude temperatures between 15° C. and 95° C. In another embodiment,controlled temperatures include temperatures between 15° C. and 70° C.Preferred temperatures are between 20° C. and 55° C., more preferablybetween 30° C. and 50° C.

[0064] Microorganisms can be cultured (e.g., maintained and/or grown) inliquid media and preferably are cultured, either continuously orintermittently, by conventional culturing methods such as standingculture, test tube culture, shaking culture (e.g., rotary shakingculture, shake flask culture, etc.), aeration spinner culture, orfermentation. In a preferred embodiment, the microorganisms are culturedin shake flasks. In a more preferred embodiment, the microorganisms arecultured in a fermentor (e.g., a fermentation process). Fermentationprocesses of the present invention include, but are not limited to,batch, fed-batch and continuous processes or methods of fermentation.The phrase “batch process” or “batch fermentation” refers to a closedsystem in which the composition of media, nutrients, supplementaladditives and the like is set at the beginning of the fermentation andnot subject to alteration during the fermentation, however, attempts maybe made to control such factors as pH and oxygen concentration toprevent excess media acidification and/or microorganism death. Thephrase “fed-batch process” or “fed-batch” fermentation refers to a batchfermentation with the exception that one or more substrates orsupplements are added (e.g., added in increments or continuously) as thefermentation progresses. The phrase “continuous process” or “continuousfermentation” refers to a system in which a defined fermentation mediais added continuously to a fermentor and an equal amount of used or“conditioned” media is simultaneously removed, preferably for recoveryof the desired product (e.g., HMBPA). A variety of such processes havebeen developed and are well-known in the art.

[0065] The phrase “culturing under conditions such that a desiredcompound is produced” includes maintaining and/or growing microorganismsunder conditions (e.g., temperature, pressure, pH, duration, etc.)appropriate or sufficient to obtain production of the desired compoundor to obtain desired yields of the particular compound being produced.For example, culturing is continued for a time sufficient to produce thedesired amount of a compound (e.g., HMBPA). Preferably, culturing iscontinued for a time sufficient to substantially reach suitableproduction of the compound (e.g., a time sufficient to reach a suitableconcentration of HMBPA or suitable ratio of HMBPA:pantothenate). In oneembodiment, culturing is continued for about 12 to 24 hours. In anotherembodiment, culturing is continued for about 24 to 36 hours, 36 to 48hours, 48 to 72 hours, 72 to 96 hours, 96 to 120 hours, 120 to 144hours, or greater than 144 hours. In yet another embodiment,microorganisms are cultured under conditions such that at least about 5to 10 g/L of compound are produced in about 36 hours, at least about 10to 20 g/L compound are produced in about 48 hours, or at least about 20to 30 g/L compound in about 72 hours. In yet another embodiment,microorganisms are cultured under conditions such that at least a ratioof HMBPA:HMBPA+pantothenate of 1:10 is achieved (i.e., 10% HMBPA versus90% pantothenate, for example, as determined by comparing the peaks whena sample of product is analyzed be HPLC), preferably such that at leasta ratio of 2:10 is achieved (20% HMBPA versus 90% pantotheante), morepreferably such that a ratio of at least 2.5:10 is achieved (25% HMBPAversus 75% pantotheante), more preferably at least 3:10 (30% HMBPAversus 70% pantotheante), 4:10 (40% HMBPA versus 60% pantotheante), 5:10(50% HMBPA versus 50% pantotheante), 6:10 (60% HMBPA versus 40%pantotheante), 7:10 (70% HMBPA versus 30% pantotheante), 8:10 (80% HMBPAversus 20% pantotheante), 9:10 (90% HMBPA versus 10% pantotheante) orgreater.

[0066] The methodology of the present invention can further include astep of recovering a desired compound (e.g., HMBPA). The term“recovering” a desired compound includes extracting, harvesting,isolating or purifying the compound from culture media. Recovering thecompound can be performed according to any conventional isolation orpurification methodology known in the art including, but not limited to,treatment with a conventional resin (e.g., anion or cation exchangeresin, non/ionic adsorption resin, etc.), treatment with a conventionaladsorbent (e.g., activated charcoal, silicic acid, silica gel,cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g.,with a conventional solvent such as an alcohol, ethyl acetate, hexaneand the like), dialysis, filtration, concentration, crystallization,recrystallization, pH adjustment, lyophilization and the like. Forexample, a compound can be recovered from culture media by firstremoving the microorganisms from the culture. Media are then passedthrough or over a cation exchange resin to remove cations and thenthrough or over an anion exchange resin to remove inorganic anions andorganic acids having stronger acidities than the compound of interest.The resulting compound can subsequently be converted to a salt (e.g., acalcium salt) as described herein.

[0067] Preferably, a desired compound of the present invention is“extracted”, “isolated” or “purified” such that the resultingpreparation is substantially free of other media components (e.g., freeof media components and/or fermentation byproducts). The language“substantially free of other media components” includes preparations ofthe desired compound in which the compound is separated from mediacomponents or fermentation byproducts of the culture from which it isproduced. In one embodiment, the preparation has greater than about 80%(by dry weight) of the desired compound (e.g., less than about 20% ofother media components or fermentation byproducts), more preferablygreater than about 90% of the desired compound (e.g., less than about10% of other media components or fermentation byproducts), still morepreferably greater than about 95% of the desired compound (e.g., lessthan about 5% of other media components or fermentation byproducts), andmost preferably greater than about 98-99% desired compound (e.g., lessthan about 1-2% other media components or fermentation byproducts). Whenthe desired compound has been derivatized to a salt, the compound ispreferably further free of chemical contaminants associated with theformation of the salt. When the desired compound has been derivatized toan alcohol, the compound is preferably further free of chemicalcontaminants associated with the formation of the alcohol.

[0068] In an alternative embodiment, the desired compound is notpurified from the microorganism, for example, when the microorganism isbiologically non-hazardous (e.g., safe). For example, the entire culture(or culture supernatant) can be used as a source of product (e.g., crudeproduct). In one embodiment, the culture (or culture supernatant) isused without modification. In another embodiment, the culture (orculture supernatant) is concentrated. In yet another embodiment, theculture (or culture supernatant) is dried or lyophilized.

[0069] Preferably, a production method of the present invention resultsin production of the desired compound at a significantly high yield. Thephrase “significantly high yield” includes a level of production oryield which is sufficiently elevated or above what is usual forcomparable production methods, for example, which is elevated to a levelsufficient for commercial production of the desired product (e.g.,production of the product at a commercially feasible cost). In oneembodiment, the invention features a production method that includesculturing a recombinant microorganism under conditions such that thedesired product (e.g., HMBPA) is produced at a level greater than 2 g/L.In another embodiment, the invention features a production method thatincludes culturing a recombinant microorganism under conditions suchthat the desired product (e.g., HMBPA) is produced at a level greaterthan 10 g/L. In another embodiment, the invention features a productionmethod that includes culturing a recombinant microorganism underconditions such that the desired product (e.g., HMBPA) is produced at alevel greater than 20 g/L. In yet another embodiment, the inventionfeatures a production method that includes culturing a recombinantmicroorganism under conditions such that the desired product (e.g.,HMBPA) is produced at a level greater than 30 g/L. In yet anotherembodiment, the invention features a production method that includesculturing a recombinant microorganism under conditions such that thedesired product (e.g., HMBPA) is produced at a level greater than 40g/L. The invention further features a production method for producingthe desired compound that involves culturing a recombinant microorganismunder conditions such that a sufficiently elevated level of compound isproduced within a commercially desirable period of time.

[0070] Depending on the biosynthetic enzyme or combination ofbiosynthetic enzymes manipulated, it may be desirable or necessary toprovide (e.g., feed) microorganisms of the present invention at leastone biosynthetic precursor such that the desired compound or compoundsare produced. The term “biosynthetic precursor” or “precursor” includesan agent or compound which, when provided to, brought into contact with,or included in the culture medium of a microorganism, serves to enhanceor increase biosynthesis of the desired product. In one embodiment, thebiosynthetic precursor or precursor is aspartate. In another embodiment,the biosynthetic precursor or precursor is β-alanine. The amount ofaspartate or β-alanine added is preferably an amount that results in aconcentration in the culture medium sufficient to enhance productivityof the microorganism (e.g., a concentration sufficient to enhanceproduction of HMBPA. The term “excess β-alanine” includes β-alaninelevels increased or higher that those routinely utilized for culturingthe microorganism in question. For example, culturing the Bacillusmicroorganisms described in the instant Examples is routinely done inthe presence of about 0-5 g/L β-alanine. Accordingly, excess β-alaninelevels can include levels of about 5-10 g/L or more preferably about5-20 g/L β-alanine. Biosynthetic precursors of the present invention canbe added in the form of a concentrated solution or suspension (e.g., ina suitable solvent such as water or buffer) or in the form of a solid(e.g., in the form of a powder). Moreover, biosynthetic precursors ofthe present invention can be added as a single aliquot, continuously orintermittently over a given period of time.

[0071] In yet another embodiment, the biosynthetic precursor is valine.In yet another embodiment, the biosynthetic precursor isα-ketoisovalerate. Preferably, valine or α-ketoisovalerate is added inan amount that results in a concentration in the medium sufficient forproduction of the desired product (e.g., HMBPA) to occur. The term“excess α-KIV” includes α-KIV levels increased or higher that thoseroutinely utilized for culturing the microorganism in question. Forexample, culturing the Bacillus microorganisms described in the instantExamples can be done in the presence of about 0-5 g/L α-KIV.Accordingly, excess α-KIV levels can include levels of about 5-10 g/L,and more preferably about 5-20 g/L. The term “excess valine” includesvaline levels increased or higher that those routinely utilized forculturing the microorganism in question. For example, culturing theBacillus microorganisms described in the instant Examples is routinelydone in the presence of about 0-0.5 g/L valine. Accordingly, excessvaline levels can include levels of about 0.5-5 g/L, preferably about5-20 g/L valine. Biosynthetic precursors are also referred to herein as“supplemental biosynthetic substrates”.

[0072] Moreover, certain aspects of the present invention includeculturing microorganisms (e.g., recombinant microorganisms) underconditions of increased steady state glucose, decreased steady statedissolved oxygen and/or decreased serine. The term “increased steadystate glucose” includes steady state glucose levels increased or higherthat those routinely utilized for culturing the microorganism inquestion. For example, culturing the Bacillus microorganisms describedin the instant Examples is routinely done in the presence of about0.2-1.0 g/L steady state glucose. Accordingly, increased steady stateglucose levels can include levels of about 1-2 g/l, about 2-5 g/l, andpreferably about 5-20 g/L steady state glucose. The term “decreasedsteady state dissolved oxygen” includes steady state dissolved oxygenlevels less or lower that those routinely utilized for culturing themicroorganism in question and, for example, inversely correlates withincreased steady state glucose levels. For example, culturing theBacillus microorganisms described in the instant Examples is routinelydone in the presence of about 10-30% dissolved oxygen. Accordingly,decreased steady state dissolved oxygen can include levels of about0-10%, and preferably about 0-5% steady state dissolved oxygen. The term“reduced serine” includes serine levels within the lower range of thoseroutinely utilized for culturing the microorganism in question. Forexample, culturing the Bacillus microorganisms described in the instantExamples is routinely done in the presence of about 0-0.5 g/L serine.Accordingly, reduced serine levels can include, for example, levels of0-0.1 g/L serine.

[0073] Another aspect of the present invention includesbiotransformation processes which feature the recombinant microorganismsdescribed herein. The term “biotransformation process”, also referred toherein as “bioconversion processes”, includes biological processes whichresults in the production (e.g., transformation or conversion) ofappropriate substrates and/or intermediate compounds into a desiredproduct (e.g., HMBPA).

[0074] In one embodiment, the invention features a biotransformationprocess for the production of HMBPA comprising contacting amicroorganism which overexpresses a reductase (e.g., overexpressesPanE1, PanE2 and/or IlvC) with appropriate substrates or precursorsunder conditions such that HMBPA is produced and recovering said HMBPA.In another embodiment, the invention features a biotransformationprocess for the production of HMBPA comprising contacting amicroorganism which has a reduced or deleted PanB activity withappropriate substrates or precursors under conditions such that HMBPA isproduced and recovering said HMBPA. In yet another embodiment, theinvention features a biotransformation process for the production ofHMBPA comprising contacting a microorganism which overexpresses at leastone reductase and has a reduced or deleted PanB activity withappropriate substrates or precursors under conditions such that HMBPA isproduced and recovering said HMBPA. Preferred recombinant microorganismsfor carrying out the above-described biotransformations include therecombinant microorganisms described herein. In yet another embodiment,the invention features a biotransformation reaction that includescontacting αHIV and β-alanine with isolated or purified PanC underconditions such that HMBPA is produced. α-HIV can optionally be obtainedby contacting α-KIV with purified or isolated reductase (e.g., PanE1,PanE2 and/or IlvC) and a source of reducing equivalent, for example,NADH. Conditions under which α-HIV or HMBPA are produced can include anyconditions which result in the desired production of α-HIV or HMBPA,respectively. In yet another embodiment, the present invention includesa method of producing α-HIV that includes culturing a microorganism thatoverexpresses PanE1 and/or PanE2, and/or IlvC and has a reduced ordeleted PanC or PanD (to reduce HMBPA or β-alanine sunthesis,respectively) under conditions such that α-HIV is produced.

[0075] The microorganism(s) and/or enzymes used in the biotransformationreactions are in a form allowing them to perform their intended function(e.g., producing a desired compound). The microorganisms can be wholecells, or can be only those portions of the cells necessary to obtainthe desired end result. The microorganisms can be suspended (e.g., in anappropriate solution such as buffered solutions or media), rinsed (e.g.,rinsed free of media from culturing the microorganism), acetone-dried,immobilized (e.g., with polyacrylamide gel or k-carrageenan or onsynthetic supports, for example, beads, matrices and the like), fixed,cross-linked or permeablized (e.g., have permeablized membranes and/orwalls such that compounds, for example, substrates, intermediates orproducts can more easily pass through said membrane or wall).

[0076] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

EXAMPLES Example I Discovery and Characterization of the[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA)Biosynthetic Pathway

[0077] In developing Bacillus strains for the production ofpantothenate, various genetic manipulations were made to enzymesinvolved in the pantothenate biosynthetic pathway and theisoleucine-valine (ilv) pathway (FIG. 1) as described in U.S. patentapplication Ser. No. 09/400,494 and U.S. patent application Ser. No.09/667,569. For example, strains having a deregulated panBCD operonand/or having deregulated panE1 exhibited enhanced pantothenateproduction (when cultured in the presence of β-alanine and α-KIV).Strains further deregulated for ilvBNC and ilvD exhibited enhancedpantothenate production in the presence of only β-alanine. Moreover, itwas possible to achieve β-alanine independence by further deregulatingpanD.

[0078] An exemplary strain is PA824, a tryptophan prototroph, Spec andTet resistant, deregulated for panBCD at the panBCD locus, deregulatedfor panE1 at the panE1 locus (two genes in the B. subtilis genome arehomologous to E. coli panE, panE1 and panE2, the former encoding themajor ketopantoate reductase involved in pantothenate production, whilepanE2 does not contribute to pantothenate synthesis (U.S. patentapplication Ser. No. 09/400,494), deregulated for ilvD at the ilvDlocus, overexpressing an ilvBNC cassette at the amyE locus, andoverexpressing panD at the bpr locus.

[0079] The production of pantothenic acid by PA824 was investigated in14 L fermentor vessels. The composition of the batch and feed media areas follows. BATCH MATERIAL g/L (final) 1 Yeast extract 10 2 Na Glutamate5 3 (NH₄)₂SO₄ 8 4 KH₂PO₄ 5 5 K₂HPO₄ 7.6

[0080] Addded After Sterilization and Cool Down 1 Glucose 2.5 2 CaCl₂0.1 3 MgCl₂ 1 4 Sodium Citrate 1 5 FeSO₄.7 H₂O 0.01 5 SM-1000X 1 ml

[0081] The final volume of the batch medium is 6 L. The trace elementsolution Sm-1000X has following composition: 0.15 g Na₂MoO₄.2H₂O, 2.5 gH₃BO₃, 0.7 g CoCl₂.6 H₂O, 0.25 g CuSO₄.5H₂O, 1.6 g MnCl₂.4H₂O, 0.3 gZnSO₄.7H₂O are dissolved in water final volume 1L).

[0082] The batch medium was inoculated with 60 ml of shake flask PA824culture (OD=10 in SVY medium: Difco Veal Infusion broth 25 g, DifcoYeast extract 5 g, Sodium Glutamate 5 g, (NH₄)₂SO₄ 2.7 g in 740 ml H₂O,autoclave; add 200 ml sterile 1 M K₂HPO₄ (pH 7) and 60 ml sterile 50%Glucose solution (final volume 1L)). The fermentation was run at 43° C.at an air flow rate of 12 L/min as a glucose limited fed batch. Theinitial batched glucose (2.5 g/L) was consumed during exponentialgrowth). Afterwards glucose concentrations were maintained between 0.2-1g/L by continuous feeding of FEED solution as follows. FEED MATERIAL g/L(final) 1 Glucose 550 2 CaCl₂ 0.1 3 SM-1000X 3 ml

[0083] The variable feed rate pump was computer controlled and linked tothe glucose concentration in the tank by an algorithm. In this examplethe total feeding was 6 L.

[0084] During fermentation the pH was set at 7.2. Control was achievedby pH measurements linked to computer control. The pH value wasmaintained by feeding either a 5% NH₃-solution or a 20% H₃PO₄-solution.NH₃ acts simultaneousely as a N-source for the fermentation. Thedissolved oxygen concentration [pO₂] was set at 30% by regulation of theagitation and aeration rate. Foaming was controlled by addition ofsilicone oil. After the stop of the addition of the feed solution, inthis example after 48 h, the fermentation was continued until the [pO₂]value reached 95%. Then the fermentation was stopped by killing themicroorganism through sterilization for 30 min. The successfulsterilization was proven by plating a sample of the fermentation brothon agar plates. The pantothenate titer in the fermentation broth was21.7 g/L after sterilization and removal of the cells by centrifugation(determined by HPLC analysis).

[0085] For HPLC analysis the fermentation broth sample was diluted withsterile water (1:40). 5 μl of this dilution was injected into a HPLCcolumn (Aqua C18, 5 μm, 150*2.0 mm, Phenomenex™). Temperature of thecolumn was held at 40° C. Mobile phase A was 14.8 mM H₃PO₃, mobile phaseB 100% Acetonitrile. Flow rate was constant at 0.5 mL/min. A gradientwas applied: start: 2% mobile phase B 0-3 min linear increase to 3%mobile phase B 3-3.5 min linear increase to 20% mobile phase B

[0086] The detection was carried out by an UV-detector (210 nm). Runtime was 7 min with an additional 3 min posttime. The retention time forpantothenic acid is 3.9 minutes. The HPLC chromatogram for the abovementioned sample is given in FIG. 4.

[0087] Identification of Compound Related to Peak with Retention Time4.7 Minutes

[0088] Under the described fermentation conditions, PA824 routinelyyields approximately 20-30 g/L pantothenate. In addition to producingsignificant quantities of pantothenate, it was discovered a secondcompound eluted with an approximate retention time of 4.7 minutes inthis system. The second prominent product formed in the fermentation wasshown to be [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid(HMBPA) (also referred to herein as “β-alanine2-(R)-iydroxyisolvalerate”, “β-alanine 2-hydroxyisolvalerate”, and/or“β-alanyl-α-hydroxyisovalarate). It was identified by its mass spectrum(FIG. 5; relative monoisotopic mass 189), ¹H- and 13C-NMR (data notshown) after chromatographic purification by reverse phase flashchromatography (mobile phase 10 mM KH₂PO₄, with increasing contents ofacetonitrile (1-50%)).

[0089] In order to verify the identity of the compound, deliberatesynthesis of racemic β-alanine 2-hydroxyisolvalerate was performed asfollows. β-alanine (2.73 g/30 mmol) and sodium methoxide (5.67 g of a30% solution in methanol/31.5 mmol) were dissolved in methanol (40 mL).Methyl 2-hydroxyisovalerate (2-hydroxy-3-methylbutyric acid methylester) (3.96 g/30 mmol) was added and refluxed for 18 hours. Methanolwas then removed by rotavap and replaced by tert-butanol (50 mL).Potassium tert-butoxide was added (50 mg) and refluxed for 26 hours. Thesolvent was removed in vacuo, the residue dissolved in water (50 mL) andpassed through a strongly acidic ion-exchange resin (H+-form Lewatite™ S100 G1; 100 mL). More water is used to rinse the ion exchanger. Theaqueous eluates are combined and the water removed in vacuo. The residueis subjected to flash chromatography (silica gel; 2% acetic acid inethyl acetate as eluent) and the product fractions evaporated to give asolid residue. The residue was recrystallized from ethyl acetate/toluene(10 mL /20 mL, respectively) and analytically pure HMBPA (β-alanine2-hydroxyisolvalerate) was obtained, which showed a relativemonoisotopic mass of 190 (189+H⁺) in the mass spec and the same ¹H-NMRresonances as the product obtained from fermentation.

[0090] The biosynthetic pathway resulting in HMBPA production is setforth in FIG. 2. The chemical structure of[R]-3-(2-hydroxy-3-methyl-butyrylamino)propionic acid (HMBPA) isdepicted in FIG. 3. As depicted in FIG. 2, HMBPA is the condensationproduct of α-hydroxyisovaleric acid (α-HIV) and β-alanine, catalyzed bythe PanC enzyme. α-HIV is generated by reduction of α-KIV, a reactionwhich is catalyzed by the reductases PanE (e.g., PanE1 and/or PanE2)and/or IlvC.

[0091] Based on the chemical structure and biosynthetic pathway leadingto HMBPA production, the present inventors formulated the followingmodel to describe the interaction between the-previously knownpantothenate and isoleucine-valine (ilv) pathways and the newlycharacterized HMBPA biosynthetic pathway. In at least one aspect, themodel states that there exist at least two pathways in microorganismsthat compete for α-KIV, the substrate for the biosynthetic enzyme PanB,namely the pantothenate biosynthetic pathway and the HMBPA biosyntheticpathway. (A third and fourth pathway competing for α-KIV are thoseresulting in the production of valine or leucine from α-KIV, see e.g.,FIG. 1). At least the pantothenate biosynthetic pathway and the HMBPAbiosynthetic pathway further produce competitive substrates for theenzyme PanC, namely α-HIV and pantoate. The model predicts that reducingPanB activity will increase α-KIV availability for α-HIV synthesis (andultimately, HMBPA synthesis) and decrease the amount of pantoate and/orpantothenate synthesized by a microorganism. Conversely, increasing PanBactivity will increase pantoate and ketopantoate availability forpantoate/pantothenate synthesis. The following examples provideexperimental support for the model and further exemplify processes forincreasing the production of HMBPA based on the model.

Examples II-VI

[0092] For Examples II-VI, quanitation of pantothenate and/or HMBPA wasperformed as follows. Aliquots of fermentation media were diluted 1:100and aliquots of test tube cultures were diluted 1:10 in water or 5%acetonitrile prior to injection on a Phenomenex Aqua™ 5μ C18 HPLC column(250×4.60 mm, 125A). Mobile phases were A=5% acetonitrile, 50 mMmonosodium phosphate buffer adjusted to pH 2.5 with phosphoric acid; andB=95% acetonitrile, 5% H₂O.

[0093] Linear gradients were as follows. Minutes Solvent A Solvent B 0100% 0% 16 100% 0% 17 0% 100% 20 0% 100% 21 100% 0%

[0094] Additional parameters and apparatus were as follows: Flowrate=1.0 ml/min; Injection volume=20 μl; Detector=Hewlett Packard 1090series DAD UV detector-3014, Signal A=197 nm, ref.=450 nm, Firmwarerevision E; Column heater=Oven tempature 40° C.; Hardware=HewlettPackard Kayak™ XA; and Software=Hewlett Packard Chemstation Plus™ familyrevision A.06.03[509].

[0095] Under these fermentation conditions, PA824 routinely yieldsapproximately 30-40 g/L pantothenate. HMBPA elutes at approximately 13minutes in this system.

Example II Ketopantoate Reductase Contributes to the Production of HMBPAand Increasing Ketopantoate Reductase Activity in Bacillus Results inEnhanced HMBPA Production

[0096] As described in Example I, a novel HPLC peak corresponding toHMBPA was observed in microorganisms overexpressing panE1 indicatingthat increased ketopantoate reductase contributes to the production ofHMBPA (in addition to production of pantothenate). As mentionedpreviously, two genes in the B. subtilis genome are homologous to the E.coli panE gene encoding ketopantoate reductase and have been named panE1and panE2. In Bacillus, the panE1 gene encodes the major ketopantoatereductase involved in pantothenate production, while panE2 does notcontribute to pantothenate synthesis. In fact, overexpression of panE2from a P₂₆ promoter leads to a reduction in pantothenate titer (seee.g., U.S. patent application Ser. No. 09/400,494).

[0097] Accordingly, it was tested whether, beside being produced by thepanE1 gene product, it was possible that a significant portion of theα-HIV necessary to make HMBPA was being produced by the panE2 geneproduct. It was hypothesized that the panE2 gene product is an enzymethat can reduce α-KIV to α-HIV, but that can not significantly reduceketopantoate to pantoate.

[0098] To test the hypothesis, panE2 was deleted from pantotheniateproduction strain PA824 (described in Example I) by transforming with aΔpanE2::cat cassette from chromosomal DNA of strain PA248 (ΔpanE2::cat)(set forth as SEQ ID NO:24, for construction see e.g., U.S. patentapplication Ser. No. 09/400,494) to give strain PA919. Three isolates ofPA919 were compared to PA824 for pantothenate and HMBPA production intest tube cultures grown in SVY plus β-alanine. TABLE 1 Production ofpantothenate and HMBPA by derivatives of PA824 and PA880 grown at 43° C.in 48 hour test tube cultures of SVY glucose + β-alanine⁵. Strain newtrait parent OD₆₀₀ [pan ] g/l [HMBPA] g/l PA824 — 13.9 4.3 0.64 PA919-1ΔpanE2::cat PA824 13.2 4.2 0.15 PA919-2 ″ ″ 14.8 3.8 0.13 PA919-3 ″ ″18.0 5.5 0.14

[0099] As indicated by the data in Table 1, all three isolates of PA919produced about four-fold lower HMBPA than PA824 demonstrating that thepanE2 gene product is a potent contributor to HMBPA synthesis. Moreover,significant increases in HMBPA production can be achieved simply byoverexpression of panE2. An exemplary plasmid for the overexpression ofpanE2, named pAN238, is set forth as SEQ ID NO:25 (FIG. 10).

Example III Increasing Production of HMBPA by Reducing PanB Activity inMicroorganisms

[0100] Strains derived from PA365 (the RL-1 lineage equivalent of PA377,described in U.S. patent application Ser. No. 09/667,569) which aredeleted for the P₂₆ panBCD cassette and which contain a P₂₆panC*Dcassette amplified at the vpr locus and either the wild type P₂₆panBcassette (PA666) or a P26 ΔpanB cassette (PA664) amplified at the bprlocus were constructed as follows. An alignment of the C-terminal aminoacids of known or suspected PanB proteins is shown in FIG. 6. Threeregions called 1, 2 and 3, that were identified having conserved orsemi-conserved amino acid residues, are indicated by arrows at the topof the figure. The B. subtilis PanB protein (RBS02239) is underlined.Two of the PanB proteins (RCY14036 and CAB56202.1) are missing region 3while the latter PanB protein is also missing region 2 and hasnon-conserved amino acid residues occupying region 1.

[0101]B. subtilis PanB variants were created that were missing regions1, 2 and 3. The desired variants were created by designing 3′ PCRprimers to amplify the B. subtilis pan B gene such that region 3,regions 2 and 3, or all three regions would be missing from the finalproduct. The PCR products were generated and cloned into E. coliexpression vector pASK-1BA3, creating plasmids pAN446, pAN447, andpAN448, respectively. The plasmids were then transformed into E. colistrain SJ2 that contains the panB6 mutation to test for complementation.Only pAN446, which is missing region 3, was able to complement. Thisindicates that region 3 is not essential for B. subtilis PanB activitybut that region 2 is required for activity or stability.

[0102] The next step in this analysis was to transfer the panB gene frompAN446 to a B. subtilis expression vector and then introduce it into astrain appropriate for testing activity of the encoded PanB protein inB. subtilis. To do this, a strain that is deleted for the P₂₆ panBCDoperon was first created. This was accomplished by first inserting a catgene between the BseRI site located just upstream of the panB RBS andthe Bg/II site located in panD, creating plasmid pAN624 (FIG. 7). Thesequence of pAN624 is set forth as SEQ ID NO:20. The resultingdeletion-substitution mutation (ΔpanBCD::cat624), which removes all ofpanB and panC, was crossed into PA354 by transformation, with selectionfor resistance to chloramphenicol on plates supplemented with 1 mMpantothenate. One of the transformants was saved and named PA644.Chromosomal DNA isolated from PA644 was analyzed by PCR and was shown tocontain the deletion-substitution mutation. As expected, PA644 requirespantothenate for growth but retains the engineered ilv genes (P₂₆ilvBNCP₂₆ilvD) as well as the P₂₆pan E1 gene originally present in PA354.Thus, it has all the enzymes involved in pantoate synthesis overproducedexcept PanB. The gene containing the shortest panB deletion was insertedinto B. subtilis expression vector pOTP61 (described in U.S. patentapplication Ser. No. 09/667,569), creating plasmid pAN627. At the sametime, a wild-type panB control gene was inserted into pOTP61, creatingplasmid pAN630. The NotI fragments of each plasmid, lacking E. colivector sequences, were ligated and transformed into PA644, withselection for resistance to tetracycline.

[0103] One transformant from each transformation was saved and furthertransformed with chromosomal DNA from PA628 with selection for Pan*.PA628 contains a multicopy P₂₆panC*D expression plasmid (pAN620)integrated at the vpr locus. In order to determine the effects of thepanB gene mutation directly on pantothenate production, plasmid pAN620,set forth as SEQ ID NO:21 and illustrated schematically in FIG. 8,provides the remaining two enzymes required for pantothenate synthesis(PanC and PanD). Four transformants from each transformation wereisolated, grown in SVY medium containing 10 g/L aspartate for 48 hours,then assayed for pantothenate production. Transformants with the3′deleted panB gene were named PA664 and those containing the wild-typegene were called PA666. The data showed that the 3′ deleted panB gene inPA664 encodes a PanB protein with greatly reduced activity. To test forHMBPA production, test tube cultures of PA365, PA666, and PA664 weregrown in SVY+aspartate medium with and without added α-KIV or pantoatefor 48 hours and then assayed for HMBPA and pantothenate as describedpreviously. TABLE 2 Effect of PanB activity and addition of precursorson HMBPA and pantothenate production, 48 hour test tube culture data,SVY + aspartate (10 g/L) medium. +α-KIV +pantoate no additions (5 g/L)(5 g/L) pan C*D pauB [pan] HMBPA [pan] HMBPA [pan] HMBPA Strain panoperon plasmid plasmid (g/L) peak* (g/L) peak (g/L) peak PA365 P₂₆panBCDNONE NONE 3.0 0.71 3.2 1.28 4.8 0.38 PA666 ΔpanBCD::cat pAN620 pAN6303.7 0.55 3.3 1.70 5.2 0.26 PA664 ΔpanBCD::cat pAN620 pAN627 0.3 1.39 0.61.76 2.5 0.74

[0104] The data presented in Table 2 demonstrate that in the absence ofsupplements, PA664 produced the most HMBPA while PA666 produced theleast, indicating an inverse correlation between PanB activity and HMBPAproduction. This is consistent with the model which predicts that thetwo pathways compete for α-KIV, the substrate for PanB, and producecompetitive substrates for PanC; lowering PanB activity would beexpected to increase α-KIV availability for α-HIV synthesis and increaseHMBPA production, correspondingly decreasing the amount of pantoatesynthesized. When α-KIV is added to the medium, all three strainsproduced significantly more HMBPA. This result evidences that α-KIV is aprecursor to HMBPA, as described in FIG. 2, and that excess α-KIV favorsHMBPA production. This result also suggests that synthesis of HMBPA isat least partially due to an overflow effect of excess α-KIV production.When pantoate was added to the medium, HMBPA was reduced by roughly 50percent in all three strains. Conversely, the strains each producedsignificantly more pantothenate. This result is also consistent with themodel that the two pathways produce competing substrates for PanC (α-HIVand pantoate). Taken together, the above results further indicate thatdecreasing pantoate synthesis should be beneficial in promoting HMBPAproduction as well as reducing pantothenate levels.

Example IV Methods for Regulating HBPA:Pantothenate Levels

[0105] As demonstrated in Examples I and II, PanE1 and/or PanE2contribute to enhanced HMBPA production as does reduced PanB activity.This Example demonstrates that overexpressing PanE1 increases HMBPAproduction relative to pantothenate production whereas overexpressingPanB decreases HMBPA production relative to pantothenate production.Furthermore, in strains overexpressing IlvC, HMBPA production isenhanced.

[0106] PA668 is a derivative of PA824 that contains extra copies ofP₂₆panB amplified at the vpr or panB locus. PA668 was constructed usingthe panB expression vector (pAN636) which allows for selection ofmultiple copies using chloramphenicol. The sequence of pAN636 is setforth as SEQ ID NO:22 and the vector is depicted schematically in FIG.9. The pAN636 NotI restriction fragment, missing the E. coli vectorsequences, was ligated and then used to transform PA824 with selectionon plates containing 5 μg/ml chloramphenicol. Transformants resistant to30 μg/ml chloramphenicol were isolated and screened for pantothenateproduction in 48 hour test tube cultures. The isolates shown produceless HMBPA that PA824 (conversely producing about 10 percent morepantothenate than PA824). A second strain, called PA669, was constructedwhich is PA824 with extra copies of P26panE1 amplified at the vpr orpanE1 locus. Strain PA669 was constructed by transforming PA824 with theself-ligated NotI fragment of plasmid pAN637 with selection forresistance to chloramphenicol. The sequence of pAN637 is set forth asSEQ ID NO:23 and the vector is depicted schematically in FIG. 10. Twoisolates of PA669 were chosen for further study; PA669-5 produces lessPanE1 than PA669-7 as judged by SDS-PAGE analysis of total cell extractsmade from the two strains.

[0107] Test tube cultures of strains PA824, PA668-2, PA668-24, and thetwo isolates of PA669 (PA669-5 and PA669-7) were grown in threedifferent media (SVY, SVY+aspartate, and SVY+aspartate +pantoate) for 48hours and then assayed for pantothenate, HMBPA, and β-alanine (Table 3).TABLE 3 Effect of extra copies of panB and panE1 on pantothenate andHMBPA production by PA824, 48 hour test tube culture data, SV medium.+aspartate (10 g/L) & no additions +aspartate (10 g/L) pantoate (5 g/L)panB panE [pan] [β-ala] HMBPA [pan] [β-ala] [pan] [β-ala] Strain plasmidplasmid (g/L) (g/L) * (g/L) (g/L) HMBPA (g/L) (g/L) HMBPA PA824 NONENONE 1.8 0.05 <0.1 4.7 2.5 0.53 5.6 2.5 <0.10 PA668-2 pAN636 NONE 1.5<0.04 <0.1 5.0 1.6 <0.10 4.9 1.2 <0.10 PA668-24 pAN636 NONE 1.8 0.05<0.1 4.9 2.8 0.34 6.1 2.6 <0.10 PA669-5 NONE pAN637 1.8 0.04 <0.1 4.23.1 0.74 5.8 2.6 0.30 PA669-7 NONE pAN637 1.8 0.06 <0.1 3.7 3.2 1.41 5.22.5 0.75

[0108] None of the strains produced detectable quantities of HMBPA inSVY medium. All strains produced roughly equivalent amounts ofpantothenate and low amounts of β-alanine indicating that β-alanine islimiting for both pantothenate and HMBPA synthesis in these cultures andthat β-alanine is a precursor for both compounds. When grown inSVY+aspartate medium, the two PA669 isolates produced more HMBPA thanPA824 whereas both PA668 isolates produced less HMBPA than PA824. It isnoteworthy that the strain that produces the most PanE1 (PA669-7)produced the most HMBPA (and the least pantothenate). This suggests thathigh levels of PanE1 favor the production of HMBPA at the expense oflower pantothenate synthesis. It is also interesting that PA668-24produced more HMBPA than PA668-2, even though SDS-PAGE analysis ofextracts from the two strains showed that they produce roughlyequivalent levels of PanB. The SDS-PAGE analysis also showed thatPA668-24 makes much more IlvC than PA668-2. Based on these data, it isproposed that IlvC influences HMBPA synthesis by increasing steady statelevels of α-KIV and/or by catalyzing α-HIV formation from α-KIV, therebyaccounting for the observed shift towards production of HMBPA.

[0109] The final set of data in Table 3 shows that adding pantoate tothe growth medium decreased HMBPA production by all strains that hadpreviously produced detectable levels, e.g., by shifting synthesistowards pantothenate. This further supports the model that α-HIV andpantoate are competitive substrates for PanC.

Example V Increasing HMBPA Production by Limiting Serine Availability

[0110] It was hypothesized that the ratio of pantothenate to HMBPAproduction could also be controlled by regulating the availability ofserine or methylene tetrahydrofolate in the microorganism cultures. Inparticular, it is proposed that decreasing the availability of serinecould increase HMBPA production relative to pantothenate production,whereas increasing the availability of serine would decrease theproduction of HMBPA relative to pantothenate production. This method isbased on the understanding that the PanB substrate,methylenetetrahydrofolate is derived from serine. Thus, regulatingserine levels should effectively regulate PanB substrate levels. To testthis hypothesis, PA824 was grown in test tube cultures of SVY glucoseplus 5 g/L β-alanine and ±5 g/L serine for 48 hours at 43° C. TABLE 4Production of HMBPA and pantothenate by PA824 with and without theaddition of serine serine added at 5 g/L OD₆₀₀ [pan] g/L [HMBPA] g/L −16.3 4.9 0.84 − 14.0 4.5 0.80 + 13.1 6.4 0.56 + 12.9 6.0 0.62

[0111] As demonstrated in Table 4, addition of serine decreases thelevel of production while conversely increasing pantothenate production.At least one method of decreasing methylene tetrahydrofolate levels inorder to regulate HMBPA production levels is to decrease the activity ofserine hydroxymethyl transferase (the glyA gene product), therebydecreasing methylene tetrahydrofolate biosynthesis in appropriatelyengineered microorganisms. At least one method of decreasing serinelevels in order to regulate HMBPA production is to decrease the activityof 3-phosphoglycerate dehydrogenase (the serA gene product).

Example VI Increasing HMBPA Production by Modifying Culture Conditionsfor Recombinant Microorganisms

[0112] In at least one fermentation (Fermentation P162), levels of HMBPAn reached 35 g/L. Briefly, fermentation of strain PA824 was carried outas in Example I but utilizing PFM-155 medium formulated as follows.BATCH MATERIAL g/L (final) 1 Amberex 1003 5 2 Cargill 200/20 (soy flour)40 3 Na Glutamate 5 4 (NH₄)₂SO₄ 8 5 MgSO₄.7H₂O 1 6 MAZU DF204C 1 7 H₂Oqs to 4 L Added After Sterilization and Cool Down 1 KH₂PO₄ 10 2K₂HPO₄.3H₂O 20 3 H₂O qs to 400 ml 1 80% Glucose 20 2 CaCl₂.2H₂O 0.1 1Sodium Citrate 1 2 FeSO₄.7H₂O 0.01 3 SM-1000X 1 X FEED 1 80% Glucose 8002 CaCl₂.2H₂O 0.8 3 H₂O qs to 3500 ml Added After Sterilization and CoolDown 1 Sodium Citrate 2.0 2 FeSO₄.7H₂O 0.02 3 SM-1000X 2 X 4 GlutamateNa 5.0 5 H₂O qs to 500 ml

[0113] However, as a result of loss of process control during thefermentation, the dissolved oxygen became limiting between 16 and 17hours and glucose began to accumulate after 16 hours.

[0114] These changes in fermentation conditions produced the followingsignificant results at or after 16 hours. Namely, synthesis of HMBPAbegan to increase with a corresponding decrease in pantothenatesynthesis. In the four hour interval before 16 hours the cultureproduced 7 g/l HMBPA, four hours afterwards, 9.0 g/l. Pantothenate wasthe reverse with 10 g/l and 6.0 g/l produced between 12-16 hours and16-20 hours, respectively. Between 20 and 36 hours the average rate ofHMBPA synthesis was 1.0 gal hr. Overall, fermentation P162 produced 35g/l of HMBPA in 36 hours.

[0115] Thus, it appears that overfeeding of glucose, and/or limitationof dissolved oxygen (e.g., beginning at about 16 hours) leads to anincrease in HMBPA production. Accordingly, two methods for increasingHMBPA production (relative to pantothenate production) are to increasesteady state glucose levels and/or decrease steady state dissolvedoxygen levels.

[0116] Equivalents Those skilled in the art will recognize, or be ableto ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be encompassed by the followingclaims.

1 25 1 194 DNA Artificial Sequence Description of ArtificialSequencepromoter sequence 1 gctattgacg acagctatgg ttcactgtcc accaaccaaaactgtgctca gtaccgccaa 60 tatttctccc ttgaggggta caaagaggtg tccctagaagagatccacgc tgtgtaaaaa 120 ttttacaaaa aggtattgac tttccctaca gggtgtgtaataatttaatt acaggcgggg 180 gcaaccccgc ctgt 194 2 163 DNA ArtificialSequence Description of Artificial Sequencepromoter sequence 2gcctacctag cttccaagaa agatatccta acagcacaag agcggaaaga tgttttgttc 60tacatccaga acaacctctg ctaaaattcc tgaaaaattt tgcaaaaagt tgttgacttt 120atctacaagg tgtggtataa taatcttaac aacagcagga cgc 163 3 127 DNA ArtificialSequence Description of Artificial Sequencepromoter sequence 3gaggaatcat agaattttgt caaaataatt ttattgacaa cgtcttatta acgttgatat 60aatttaaatt ttatttgaca aaaatgggct cgtgttgtac aataaatgta gtgaggtgga 120tgcaatg 127 4 24 DNA Artificial Sequence Description of ArtificialSequenceribosome binding site 4 taaacatgag gaggagaaaa catg 24 5 28 DNAArtificial Sequence Description of Artificial Sequenceribosome bindingsite 5 attcgagaaa tggagagaat ataatatg 28 6 13 DNA Artificial SequenceDescription of Artificial Sequenceribosome binding site 6 agaaaggagg tga13 7 23 DNA Artificial Sequence Description of ArtificialSequenceribosome binding site 7 ttaagaaagg aggtgannnn atg 23 8 23 DNAArtificial Sequence Description of Artificial Sequenceribosome bindingsite 8 ttagaaagga ggtgannnnn atg 23 9 23 DNA Artificial SequenceDescription of Artificial Sequenceribosome binding site 9 agaaaggaggtgannnnnnn atg 23 10 22 DNA Artificial Sequence Description ofArtificial Sequenceribosome binding site 10 agaaaggagg tgannnnnna tg 2211 25 DNA Artificial Sequence Description of Artificial Sequenceribosomebinding site 11 ccctctagaa ggaggagaaa acatg 25 12 24 DNA ArtificialSequence Description of Artificial Sequenceribosome binding site 12ccctctagag gaggagaaaa catg 24 13 23 DNA Artificial Sequence Descriptionof Artificial Sequenceribosome binding site 13 ttagaaagga ggatttaaat atg23 14 23 DNA Artificial Sequence Description of ArtificialSequenceribosome binding site 14 ttagaaagga ggtttaatta atg 23 15 23 DNAArtificial Sequence Description of Artificial Sequenceribosome bindingsite 15 ttagaaagga ggtgatttaa atg 23 16 23 DNA Artificial SequenceDescription of Artificial Sequenceribosome binding site 16 ttagaaaggaggtgtttaaa atg 23 17 28 DNA Artificial Sequence Description ofArtificial Sequenceribosome binding site 17 attcgagaaa ggaggtgaatataatatg 28 18 27 DNA Artificial Sequence Description of ArtificialSequenceribosome binding site 18 attcgagaaa ggaggtgaat aataatg 27 19 28DNA Artificial Sequence Description of Artificial Sequenceribosomebinding site 19 attcgtagaa aggaggtgaa ttaatatg 28 20 6886 DNA ArtificialSequence Description of Artificial Sequence vector 20 aagaaaccaattgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60 tctcgctaaccaaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120 aagccatgacaaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180 attatttgcacggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240 atcctacctgacgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg 300 ctaacaggaggaattaacca tggatccgag ctcgacagta tcaagcactt cacaatctgg 360 gagctgaaagcccgccttat gtagctcata cttgacaaat ccaaggtcaa aatggatatt 420 gtgggcgacaaaataagcgc cgtcaagcaa ttggaatact tcttcagcaa ctgcttcaaa 480 tggctgttcattctcgacca tttgattaga gattccagta agctgctcaa taaaagcagg 540 gattgatttatttggattaa tgtattttga aaaccgctca gtaatttgtc cattttcgat 600 tacaaccgctgcgatttgta tgattttatc gcctttcttc ggcgaattcc ctgttgtctc 660 tacatctataacaacgaacc gttgcttatt cattaaaatg gacacctcaa ttcttgcata 720 cgacaaaagtgtaacacgtt ttgtacggaa atggagcggc aaaaccgttt tactctcaaa 780 atcttaaaagaaaacccccg ataaaggggg cttttcttct acaaaattgt acgggctggt 840 tcgttccccagcatttgttc aattttgttt tgatcattca gaacagccac tttcggctca 900 tggcttgccgcttcttgatc agacatcatt ttgtaggaaa taataatgac cttatctcct 960 tcctgcacaaggcgtgcggc tgcaccgttt aagcatatga cgccgcttcc ccgtttacca 1020 ggaataatatacgtttcaag acgtgctcca ttattattat tcacaatttg tactttttca 1080 ttaggaagcattcccacagc atcaatgaga tcctctagag tcgacctgca ggcatgcaag 1140 cttccgtcgacgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt 1200 gaggccgttgagcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag 1260 tcccccggccacggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa 1320 gtggcgagcccgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc 1380 tgtggcgccggtgatgccgg ccacgatgcg tccggcgtag aggatcaatc ttcatccatt 1440 ccaaggtaaatcccccttcg ccgtttctgt taccattata caccttttga accttaacgt 1500 aaacgttaagttttaaaaaa caataaaaaa gacgagcagc atacagcacc cgtctttcac 1560 tttcctgtttaagctaaact tcccgccact gacagagact ctttttgaag gctttcagaa 1620 agcactcgatacgcgatctg gagctgtaat ataaaaacct tcttcaacta acggggcagg 1680 ttagtgacattagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 1740 gccagtcattaggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 1800 aaccatcacaaacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 1860 tattaatgaattttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 1920 ttaagttaaacccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 1980 gtataggtgttttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 2040 ataaatcataaaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 2100 tagatacaccatcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 2160 cgtcgctattgtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 2220 aaataaatgcagggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 2280 tatcaatttctgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 2340 cttttctcttccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 2400 tttttatctaaagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 2460 ttttttaaaagtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 2520 ccaattttcgtttgttgaac taatgggtgc tttagttgaa gaataaagac cacattaaaa 2580 aatgtggtcttttgtgtttt tttaaaggat ttgagcgtag cgaaaaatcc ttttctttct 2640 tatcttgataataagggtaa ctattgcatg ataagctgtc aaacatgaga attcccgttt 2700 tcttctgcaagccaaaaaac cttccgttac aacgagaagg attcttcact ttctaaagtt 2760 cggcgagtttcatccctctg tcccagtcct tttttggatc aaggcagact gctgcaatgt 2820 ctatctattttaataatagg tgcagttcgc aggcgatact gcccaatgga agtataccaa 2880 aatcaacgggcttgtaccaa cacattagcc caattcgata tcggcagaat agattttttt 2940 aatgccttcgttcgtttcta aaagcagaac gccttcatca tctataccta acgccttacc 3000 gtaaaaggttccgtttaacg ttctggctct catattagtg ccaataccga gcgcatagct 3060 ttcccataaaagcttaatcg gcgtaaatcc gtgcgtcata taatcccggt accgtttctc 3120 aaagcatagtaaaatatgct ggatgacgcc ggcccgatca attttttccc cagcagcttg 3180 gctgaggcttgtcgcgatgt ccttcaattc atctggaaaa tcattaggct gctggttaaa 3240 cggtctccagcttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa 3300 atcagaacgcagaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt 3360 cccacctgaccccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg 3420 gtctccccatgcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga 3480 aagactgggcctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 3540 atccgccgggagcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 3600 gcccgccataaactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt 3660 ttgcgtttctacaaactctt tttgtttatt tttctaaata cattcaaata tgtatccgct 3720 catgagacaataaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 3780 tcaacatttccgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 3840 tcacccagaaacgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 3900 ttacatcgaactggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 3960 ttttccaatgatgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 4020 cgccgggcaagagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 4080 ctcaccagtcacagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 4140 tgccataaccatgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 4200 gaaggagctaaccgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 4260 ggaaccggagctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 4320 aatggcaacaacgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 4380 acaattaatagactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 4440 tccggctggctggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 4500 cattgcagcactggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 4560 gagtcaggcaactatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 4620 taagcattggtaactgtcag accaagttta ctcatatata ctttagattg atttaaaact 4680 tcatttttaatttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 4740 cccttaacgtgagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 4800 ttcttgagatcctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 4860 accagcggtggtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 4920 cttcagcagagcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 4980 cttcaagaactctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 5040 tgctgccagtggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 5100 taaggcgcagcggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 5160 gacctacaccgaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 5220 agggagaaaggcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 5280 ggagcttccagggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 5340 acttgagcgtcgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 5400 caacgcggcctttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 5460 tgcgttatcccctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 5520 tcgccgcagccgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 5580 gatgcggtattttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 5640 cagtacaatctgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 5700 gactgggtcatggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 5760 tgtctgctcccggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 5820 cagaggttttcaccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag 5880 gcgaagcggcatgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta 5940 tgctactccgtcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct 6000 acatcattcactttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat 6060 tttttaaatacccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg 6120 gcgataggcatccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg 6180 cgccagcttaagacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc 6240 gacaagcaaacatgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg 6300 ctgatgtactgacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta 6360 atcgcttccatgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa 6420 tagcgcccttccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc 6480 tggtgcgcttcatccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc 6540 cattcatgccagtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc 6600 tccggatgacgaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt 6660 cggcaaacaaattctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg 6720 agaatataacctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc 6780 tcaatcggcgttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga 6840 tcattttgcgcttcagccat acttttcata ctcccgccat tcagag 6886 21 7140 DNA ArtificialSequence Description of Artificial Sequence vector 21 tcggcggccgcttcgtcgac cgaaacagca gttataaggc atgaagctgt ccggtttttg 60 caaaagtggctgtgactgta aaaagaaatc gaaaaagacc gttttgtgtg aaaacggtct 120 ttttgtttccttttaaccaa ctgccataac tcgaggccta cctagcttcc aagaaagata 180 tcctaacagcacaagagcgg aaagatgttt tgttctacat ccagaacaac ctctgctaaa 240 attcctgaaaaattttgcaa aaagttgttg actttatcta caaggtgtgg tataataatc 300 ttaacaacagcaggacgctc tagattagaa aggaggattt aaatatgaga cagattactg 360 atatttcacagctgaaagaa gccataaaac aataccattc agagggcaag tcaatcggat 420 ttgttccgacgatggggttt ctgcatgagg ggcatttaac cttagcagac aaagcaagac 480 aagaaaacgacgccgttatt atgagtattt ttgtgaatcc tgcacaattc ggccctaatg 540 aagattttgaagcatatccg cgcgatattg agcgggatgc agctcttgca gaaaacgccg 600 gagtcgatattctttttacg ccagatgctc atgatatgta tcccggtgaa aagaatgtca 660 cgattcatgtagaaagacgc acagacgtgt tatgcgggcg ctcaagagaa ggacattttg 720 acggggtcgcgatcgtactg acgaagcttt tcaatctagt caagccgact cgtgcctatt 780 tcggtttaaaagatgcgcag caggtagctg ttgttgatgg gttaatcagc gacttcttca 840 tggatattgaattggttcct gtcgatacgg tcagagagga agacggctta gccaaaagct 900 ctcgcaatgtatacttaaca gctgaggaaa gaaaagaagc gcctaagctg tatcgggccc 960 ttcaaacaagtgcggaactt gtccaagccg gtgaaagaga tcctgaagcg gtgataaaag 1020 ctgcaaaagatatcattgaa acgactagcg gaaccataga ctatgtagag ctttattcct 1080 atccggaactcgagcctgtg aatgaaattg ctggaaagat gattctcgct gttgcagttg 1140 ctttttcaaaagcgcgttta atagataata tcattattga tattcgtaga aaggaggtga 1200 attaatatgtatcgtacgat gatgagcggc aaacttcaca gggcaactgt tacggaagca 1260 aacctgaactatgtgggaag cattacaatt gatgaagatc tcattgatgc tgtgggaatg 1320 cttcctaatgaaaaagtaca aattgtgaat aataataatg gagcacgtct tgaaacgtat 1380 attattcctggtaaacgggg aagcggcgtc atatgcttaa acggtgcagc cgcacgcctt 1440 gtgcaggaaggagataaggt cattattatt tcctacaaaa tgatgtctga tcaagaagcg 1500 gcaagccatgagccgaaagt ggctgttctg aatgatcaaa acaaaattga acaaatgctg 1560 gggaacgaaccagcccgtac aattttgtaa aggatcctgt tttggcggat gagagaagat 1620 tttcagcctgatacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 1680 tggcggcagtagcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 1740 tagcgccgatggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 1800 taaaacgaaaggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 1860 acgctctcctgagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 1920 ccggagggtggcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 1980 ccatcctgacggatggcctt tttgcgtttc tacaaactct tggtaccgag acgatcgtcc 2040 tctttgttgtagcccatcac ttttgctgaa gagtaggagc cgaaagtgac ggcgtattca 2100 ttgagcggcagctgagtcgc accgacagaa atcgcttctc ttgatgtgcc cggcgatccg 2160 actgtccagccgttcggtcc gctgttgccg tttgaggtaa cagcgacaac gccttctgac 2220 atggcccagtcaagcgctgt gcttgtcgcc cagtccgggt tgtttaaaga gtttccgaga 2280 gacaggttcatcacatctgc cccgtcctgc actgcacgtt ccacgcccgc gatgacgttt 2340 tccgttgtgccgcttccgcc aggccctaac acacgataag caagaagtgt ggcatcaggc 2400 gctacgcctttaatcgttcc gtttgcagcc acagttccgg ctacgtgtgt gccatggtca 2460 gttgcctcgcccctcggatc gccggttggt gtttcttttg gatcgtaatc attgtccaca 2520 aaatcgtatcctttatattg tccaaagttt ttcttcagat ctgggtgatt gtattcaacc 2580 ccagtgtcaataatcgccac cttgatgcct tttcctgtgt agcctaaatc ccatgcatcg 2640 tttgctccgatataaggcgc actgtcatcc atttgcggag atacggcgtc ttcggagatt 2700 gtggggaattctcatgtttg acagcttatc atgcaatagt tacccttatt atcaagataa 2760 gaaagaaaaggatttttcgc tacgctcaaa tcctttaaaa aaacacaaaa gaccacattt 2820 tttaatgtggtctttattct tcaactaaag cacccattag ttcaacaaac gaaaattgga 2880 taaagtgggatatttttaaa atatatattt atgttacagt aatattgact tttaaaaaag 2940 gattgattctaatgaagaaa gcagacaagt aagcctccta aattcacttt agataaaaat 3000 ttaggaggcatatcaaatga actttaataa aattgattta gacaattgga agagaaaaga 3060 gatatttaatcattatttga accaacaaac gacttttagt ataaccacag aaattgatat 3120 tagtgttttataccgaaaca taaaacaaga aggatataaa ttttaccctg catttatttt 3180 cttagtgacaagggtgataa actcaaatac agcttttaga actggttaca atagcgacgg 3240 agagttaggttattgggata agttagagcc actttataca atttttgatg gtgtatctaa 3300 aacattctctggtatttgga ctcctgtaaa gaatgacttc aaagagtttt atgatttata 3360 cctttctgatgtagagaaat ataatggttc ggggaaattg tttcccaaaa cacctatacc 3420 tgaaaatgctttttctcttt ctattattcc atggacttca tttactgggt ttaacttaaa 3480 tatcaataataatagtaatt accttctacc cattattaca gcaggaaaat tcattaataa 3540 aggtaattcaatatatttac cgctatcttt acaggtacat cattctgttt gtgatggtta 3600 tcatgcaggattgtttatga actctattca ggaattgtca gataggccta atgactggct 3660 tttataatatgagataatgc cgactgtact ttttacagtc ggttttctaa tgtcactaac 3720 ctgccccgttagttgaagaa cgaagcggcc gcaattcttg aagacgaaag ggcctcgtga 3780 tacgcctatttttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 3840 cttttcggggaaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 3900 tgtatccgctcatgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3960 gtatgagtattcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 4020 ctgtttttgctcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 4080 cacgagtgggttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 4140 ccgaagaacgttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 4200 cccgtattgacgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 4260 tggttgagtactcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 4320 tatgcagtgctgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 4380 tcggaggaccgaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 4440 ttgatcgttgggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 4500 tgcctgcagcaatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 4560 cttcccggcaacaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 4620 gctcggcccttccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4680 ctcgcggtatcattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4740 acacgacggggagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4800 cctcactgattaagcattgg taactgtcag accaagttta ctcatatata ctttagattg 4860 atttaaaacttcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 4920 tgaccaaaatcccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 4980 tcaaaggatcttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 5040 aaccaccgctaccagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 5100 aggtaactggcttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 5160 taggccaccacttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 5220 taccagtggctgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 5280 agttaccggataaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 5340 tggagcgaacgacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 5400 cgcttcccgaagggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 5460 agcgcacgagggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 5520 gccacctctgacttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 5580 aaaacgccagcaacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 5640 tgttctttcctgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 5700 ctgataccgctcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 5760 aagagcgcctgatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 5820 ggtgcactctcagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct 5880 atcgctacgtgactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc 5940 ctgacgggcttgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 6000 ctgcatgtgtcagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag 6060 ctcatcagcgtggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc 6120 gttgagtttctccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc 6180 ggttttttcctgtttggtca cttgatgcct ccgtgtaagg gggaatttct gttcatgggg 6240 gtaatgataccgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat 6300 gcccggttactggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag 6360 agaaaaatcactcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag 6420 ggtagccagcagcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc 6480 cgcgtttccagactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc 6540 gcagacgttttgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc 6600 taaccagtaaggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg 6660 cgcacccgtggccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg 6720 gcggacgcgatggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag 6780 aattgattggctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca 6840 ttcaggtcgaggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt 6900 atagggcggcgcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa 6960 tcgccgtgacgatcagcggt ccagtgatcg aagttaggct ggtaagagcc gcgagcgatc 7020 cttgaagctgtccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg 7080 gcatcccgatgccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg 7140 22 6725 DNAArtificial Sequence Description of Artificial Sequence vector 22tcggcggccg cttcgtcgac cgaaacagca gttataaggc atgaagctgt ccggtttttg 60caaaagtggc tgtgactgta aaaagaaatc gaaaaagacc gttttgtgtg aaaacggtct 120ttttgtttcc ttttaaccaa ctgccataac tcgaggccta cctagcttcc aagaaagata 180tcctaacagc acaagagcgg aaagatgttt tgttctacat ccagaacaac ctctgctaaa 240attcctgaaa aattttgcaa aaagttgttg actttatcta caaggtgtgg tataataatc 300ttaacaacag caggacgctc tagaaggagg agaaaacatg aaaacaaaac tggattttct 360aaaaatgaag gagtctgaag aaccgattgt catgctgacc gcttatgatt atccggcagc 420taaacttgct gaacaagcgg gagttgacat gattttagtc ggtgattcac ttggaatggt 480cgtcctcggc cttgattcaa ctgtcggtgt gacagttgcg gacatgatcc atcatacaaa 540agccgttaaa aggggtgcgc cgaatacctt tattgtgaca gatatgccgt ttatgtctta 600tcacctgtct aaggaagata cgctgaaaaa tgcagcggct atcgttcagg aaagcggagc 660tgacgcactg aagcttgagg gcggagaagg cgtgtttgaa tccattcgcg cattgacgct 720tggaggcatt ccagtagtca gtcacttagg tttgacaccg cagtcagtcg gcgtactggg 780cggctataaa gtacagggca aagacgaaca aagcgccaaa aaattaatag aagacagtat 840aaaatgcgaa gaagcaggag ctatgatgct tgtgctggaa tgtgtgccgg cagaactcac 900agccaaaatt gccgagacgc taagcatacc ggtcattgga atcggggctg gtgtgaaagc 960ggacggacaa gttctcgttt atcatgatat tatcggccac ggtgttgaga gaacacctaa 1020atttgtaaag caatatacgc gcattgatga aaccatcgaa acagcaatca gcggatatgt 1080tcaggatgta agacatcgtg ctttccctga acaaaagcat tcctttcaaa tgaaccagac 1140agtgcttgac ggcttgtacg ggggaaaata agggggggat cctgttttgg cggatgagag 1200aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat 1260ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa 1320cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca 1380tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc 1440ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca 1500acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca 1560gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttggta ccgagacgat 1620cgtcctcttt gttgtagccc atcacttttg ctgaagagta ggagccgaaa gtgacggcgt 1680attcattgag cggcagctga gtcgcaccga cagaaatcgc ttctcttgat gtgcccggcg 1740atccgactgt ccagccgttc ggtccgctgt tgccgtttga ggtaacagcg acaacgcctt 1800ctgacatggc ccagtcaagc gctgtgcttg tcgcccagtc cgggttgttt aaagagtttc 1860cgagagacag gttcatcaca tctgccccgt cctgcactgc acgttccacg cccgcgatga 1920cgttttccgt tgtgccgctt ccgccaggcc ctaacacacg ataagcaaga agtgtggcat 1980caggcgctac gcctttaatc gttccgtttg cagccacagt tccggctacg tgtgtgccat 2040ggtcagttgc ctcgcccctc ggatcgccgg ttggtgtttc ttttggatcg taatcattgt 2100ccacaaaatc gtatccttta tattgtccaa agtttttctt cagatctggg tgattgtatt 2160caaccccagt gtcaataatc gccaccttga tgccttttcc tgtgtagcct aaatcccatg 2220catcgtttgc tccgatataa ggcgcactgt catccatttg cggagatacg gcgtcttcgg 2280agattgtggg gaattctcat gtttgacagc ttatcatgca atagttaccc ttattatcaa 2340gataagaaag aaaaggattt ttcgctacgc tcaaatcctt taaaaaaaca caaaagacca 2400cattttttaa tgtggtcttt attcttcaac taaagcaccc attagttcaa caaacgaaaa 2460ttggataaag tgggatattt ttaaaatata tatttatgtt acagtaatat tgacttttaa 2520aaaaggattg attctaatga agaaagcaga caagtaagcc tcctaaattc actttagata 2580aaaatttagg aggcatatca aatgaacttt aataaaattg atttagacaa ttggaagaga 2640aaagagatat ttaatcatta tttgaaccaa caaacgactt ttagtataac cacagaaatt 2700gatattagtg ttttataccg aaacataaaa caagaaggat ataaatttta ccctgcattt 2760attttcttag tgacaagggt gataaactca aatacagctt ttagaactgg ttacaatagc 2820gacggagagt taggttattg ggataagtta gagccacttt atacaatttt tgatggtgta 2880tctaaaacat tctctggtat ttggactcct gtaaagaatg acttcaaaga gttttatgat 2940ttataccttt ctgatgtaga gaaatataat ggttcgggga aattgtttcc caaaacacct 3000atacctgaaa atgctttttc tctttctatt attccatgga cttcatttac tgggtttaac 3060ttaaatatca ataataatag taattacctt ctacccatta ttacagcagg aaaattcatt 3120aataaaggta attcaatata tttaccgcta tctttacagg tacatcattc tgtttgtgat 3180ggttatcatg caggattgtt tatgaactct attcaggaat tgtcagatag gcctaatgac 3240tggcttttat aatatgagat aatgccgact gtacttttta cagtcggttt tctaatgtca 3300ctaacctgcc ccgttagttg aagaacgaag cggccgcaat tcttgaagac gaaagggcct 3360cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg 3420tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 3480aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 3540gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 3600ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 3660gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 3720tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 3780attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 3840tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 3900agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 3960aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 4020tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 4080cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 4140tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 4200tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 4260tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 4320tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 4380aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 4440gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 4500tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 4560aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 4620aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 4680tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 4740gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 4800cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 4860acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 4920cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 4980cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 5040aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 5100gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 5160atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 5220tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 5280gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 5340agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 5400catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact 5460ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac 5520gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 5580gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg 5640taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc 5700agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta 5760agggcggttt tttcctgttt ggtcacttga tgcctccgtg taagggggaa tttctgttca 5820tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg 5880aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg 5940accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc 6000cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg 6060acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc 6120aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat 6180tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga 6240tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg 6300agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc 6360gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc 6420ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac 6480aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc 6540ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt aggctggtaa gagccgcgag 6600cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa 6660cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc 6720tcgcg 6725 23 6806 DNA Artificial Sequence Description of ArtificialSequence vector 23 tcggcggccg cttcgtcgac cgaaacagca gttataaggcatgaagctgt ccggtttttg 60 caaaagtggc tgtgactgta aaaagaaatc gaaaaagaccgttttgtgtg aaaacggtct 120 ttttgtttcc ttttaaccaa ctgccataac tcgaggcctacctagcttcc aagaaagata 180 tcctaacagc acaagagcgg aaagatgttt tgttctacatccagaacaac ctctgctaaa 240 attcctgaaa aattttgcaa aaagttgttg actttatctacaaggtgtgg tataataatc 300 ttaacaacag caggacgctc tagacaattg agatcttaagaaaggaggtg ttaattaatg 360 aagattggaa tcattggcgg aggctccgtt ggtcttttatgcgcctatta tttgtcactt 420 tatcacgacg tgactgttgt gacgaggcgg caagaacaggctgcggccat tcagtctgaa 480 ggaatccggc tttataaagg cggggaggaa ttcagggctgattgcagtgc ggacacgagt 540 atcaattcgg actttgacct gcttgtcgtg acagtgaagcagcatcagct tcaatctgtt 600 ttttcgtcgc ttgaacgaat cgggaagacg aatatattatttttgcaaaa cggcatgggg 660 catatccacg acctaaaaga ctggcacgtt ggccattccatttatgttgg aatcgttgag 720 cacggagctg taagaaaatc ggatacagct gttgatcatacaggcctagg tgcgataaaa 780 tggagcgcgt tcgacgatgc tgaaccagac cggctgaacatcttgtttca gcataaccat 840 tcggattttc cgatttatta tgagacggat tggtaccgtctgctgacggg caagctgatt 900 gtaaatgcgt gtattaatcc tttaactgcg ttattgcaagtgaaaaatgg agaactgctg 960 acaacgccag cttatctggc ttttatgaag ctggtatttcaggaggcatg ccgcatttta 1020 aaacttgaaa atgaagaaaa ggcttgggag cgggttcaggccgtttgtgg gcaaacgaaa 1080 gagaatcgtt catcaatgct ggttgacgtc attggaggccggcagacgga agctgacgcc 1140 attatcggat acttattgaa ggaagcaagt cttcaaggtcttgatgccgt ccacctagag 1200 tttttatatg gcagcatcaa agcattggag cgaaataccaacaaagtggt ttactaagga 1260 tcctgttttg gcggatgaga gaagattttc agcctgatacagattaaatc agaacgcaga 1320 agcggtctga taaaacagaa tttgcctggc ggcagtagcgcggtggtccc acctgacccc 1380 atgccgaact cagaagtgaa acgccgtagc gccgatggtagtgtggggtc tccccatgcg 1440 agagtaggga actgccaggc atcaaataaa acgaaaggctcagtcgaaag actgggcctt 1500 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagtaggacaaatc cgccgggagc 1560 ggatttgaac gttgcgaagc aacggcccgg agggtggcgggcaggacgcc cgccataaac 1620 tgccaggcat caaattaagc agaaggccat cctgacggatggcctttttg cgtttctaca 1680 aactcttggt accgagacga tcgtcctctt tgttgtagcccatcactttt gctgaagagt 1740 aggagccgaa agtgacggcg tattcattga gcggcagctgagtcgcaccg acagaaatcg 1800 cttctcttga tgtgcccggc gatccgactg tccagccgttcggtccgctg ttgccgtttg 1860 aggtaacagc gacaacgcct tctgacatgg cccagtcaagcgctgtgctt gtcgcccagt 1920 ccgggttgtt taaagagttt ccgagagaca ggttcatcacatctgccccg tcctgcactg 1980 cacgttccac gcccgcgatg acgttttccg ttgtgccgcttccgccaggc cctaacacac 2040 gataagcaag aagtgtggca tcaggcgcta cgcctttaatcgttccgttt gcagccacag 2100 ttccggctac gtgtgtgcca tggtcagttg cctcgcccctcggatcgccg gttggtgttt 2160 cttttggatc gtaatcattg tccacaaaat cgtatcctttatattgtcca aagtttttct 2220 tcagatctgg gtgattgtat tcaaccccag tgtcaataatcgccaccttg atgccttttc 2280 ctgtgtagcc taaatcccat gcatcgtttg ctccgatataaggcgcactg tcatccattt 2340 gcggagatac ggcgtcttcg gagattgtgg ggaattctcatgtttgacag cttatcatgc 2400 aatagttacc cttattatca agataagaaa gaaaaggatttttcgctacg ctcaaatcct 2460 ttaaaaaaac acaaaagacc acatttttta atgtggtctttattcttcaa ctaaagcacc 2520 cattagttca acaaacgaaa attggataaa gtgggatatttttaaaatat atatttatgt 2580 tacagtaata ttgactttta aaaaaggatt gattctaatgaagaaagcag acaagtaagc 2640 ctcctaaatt cactttagat aaaaatttag gaggcatatcaaatgaactt taataaaatt 2700 gatttagaca attggaagag aaaagagata tttaatcattatttgaacca acaaacgact 2760 tttagtataa ccacagaaat tgatattagt gttttataccgaaacataaa acaagaagga 2820 tataaatttt accctgcatt tattttctta gtgacaagggtgataaactc aaatacagct 2880 tttagaactg gttacaatag cgacggagag ttaggttattgggataagtt agagccactt 2940 tatacaattt ttgatggtgt atctaaaaca ttctctggtatttggactcc tgtaaagaat 3000 gacttcaaag agttttatga tttatacctt tctgatgtagagaaatataa tggttcgggg 3060 aaattgtttc ccaaaacacc tatacctgaa aatgctttttctctttctat tattccatgg 3120 acttcattta ctgggtttaa cttaaatatc aataataatagtaattacct tctacccatt 3180 attacagcag gaaaattcat taataaaggt aattcaatatatttaccgct atctttacag 3240 gtacatcatt ctgtttgtga tggttatcat gcaggattgtttatgaactc tattcaggaa 3300 ttgtcagata ggcctaatga ctggctttta taatatgagataatgccgac tgtacttttt 3360 acagtcggtt ttctaatgtc actaacctgc cccgttagttgaagaacgaa gcggccgcaa 3420 ttcttgaaga cgaaagggcc tcgtgatacg cctatttttataggttaatg tcatgataat 3480 aatggtttct tagacgtcag gtggcacttt tcggggaaatgtgcgcggaa cccctatttg 3540 tttatttttc taaatacatt caaatatgta tccgctcatgagacaataac cctgataaat 3600 gcttcaataa tattgaaaaa ggaagagtat gagtattcaacatttccgtg tcgcccttat 3660 tccctttttt gcggcatttt gccttcctgt ttttgctcacccagaaacgc tggtgaaagt 3720 aaaagatgct gaagatcagt tgggtgcacg agtgggttacatcgaactgg atctcaacag 3780 cggtaagatc cttgagagtt ttcgccccga agaacgttttccaatgatga gcacttttaa 3840 agttctgcta tgtggcgcgg tattatcccg tattgacgccgggcaagagc aactcggtcg 3900 ccgcatacac tattctcaga atgacttggt tgagtactcaccagtcacag aaaagcatct 3960 tacggatggc atgacagtaa gagaattatg cagtgctgccataaccatga gtgataacac 4020 tgcggccaac ttacttctga caacgatcgg aggaccgaaggagctaaccg cttttttgca 4080 caacatgggg gatcatgtaa ctcgccttga tcgttgggaaccggagctga atgaagccat 4140 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatggcaacaacgt tgcgcaaact 4200 attaactggc gaactactta ctctagcttc ccggcaacaattaatagact ggatggaggc 4260 ggataaagtt gcaggaccac ttctgcgctc ggcccttccggctggctggt ttattgctga 4320 taaatctgga gccggtgagc gtgggtctcg cggtatcattgcagcactgg ggccagatgg 4380 taagccctcc cgtatcgtag ttatctacac gacggggagtcaggcaacta tggatgaacg 4440 aaatagacag atcgctgaga taggtgcctc actgattaagcattggtaac tgtcagacca 4500 agtttactca tatatacttt agattgattt aaaacttcatttttaattta aaaggatcta 4560 ggtgaagatc ctttttgata atctcatgac caaaatcccttaacgtgagt tttcgttcca 4620 ctgagcgtca gaccccgtag aaaagatcaa aggatcttcttgagatcctt tttttctgcg 4680 cgtaatctgc tgcttgcaaa caaaaaaacc accgctaccagcggtggttt gtttgccgga 4740 tcaagagcta ccaactcttt ttccgaaggt aactggcttcagcagagcgc agataccaaa 4800 tactgtcctt ctagtgtagc cgtagttagg ccaccacttcaagaactctg tagcaccgcc 4860 tacatacctc gctctgctaa tcctgttacc agtggctgctgccagtggcg ataagtcgtg 4920 tcttaccggg ttggactcaa gacgatagtt accggataaggcgcagcggt cgggctgaac 4980 ggggggttcg tgcacacagc ccagcttgga gcgaacgacctacaccgaac tgagatacct 5040 acagcgtgag ctatgagaaa gcgccacgct tcccgaagggagaaaggcgg acaggtatcc 5100 ggtaagcggc agggtcggaa caggagagcg cacgagggagcttccagggg gaaacgcctg 5160 gtatctttat agtcctgtcg ggtttcgcca cctctgacttgagcgtcgat ttttgtgatg 5220 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaacgcggcctttt tacggttcct 5280 ggccttttgc tggccttttg ctcacatgtt ctttcctgcgttatcccctg attctgtgga 5340 taaccgtatt accgcctttg agtgagctga taccgctcgccgcagccgaa cgaccgagcg 5400 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatgcggtattttc tccttacgca 5460 tctgtgcggt atttcacacc gcatatggtg cactctcagtacaatctgct ctgatgccgc 5520 atagttaagc cagtatacac tccgctatcg ctacgtgactgggtcatggc tgcgccccga 5580 cacccgccaa cacccgctga cgcgccctga cgggcttgtctgctcccggc atccgcttac 5640 agacaagctg tgaccgtctc cgggagctgc atgtgtcagaggttttcacc gtcatcaccg 5700 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggtcgtgaagcga ttcacagatg 5760 tctgcctgtt catccgcgtc cagctcgttg agtttctccagaagcgttaa tgtctggctt 5820 ctgataaagc gggccatgtt aagggcggtt ttttcctgtttggtcacttg atgcctccgt 5880 gtaaggggga atttctgttc atgggggtaa tgataccgatgaaacgagag aggatgctca 5940 cgatacgggt tactgatgat gaacatgccc ggttactggaacgttgtgag ggtaaacaac 6000 tggcggtatg gatgcggcgg gaccagagaa aaatcactcagggtcaatgc cagcgcttcg 6060 ttaatacaga tgtaggtgtt ccacagggta gccagcagcatcctgcgatg cagatccgga 6120 acataatggt gcagggcgct gacttccgcg tttccagactttacgaaaca cggaaaccga 6180 agaccattca tgttgttgct caggtcgcag acgttttgcagcagcagtcg cttcacgttc 6240 gctcgcgtat cggtgattca ttctgctaac cagtaaggcaaccccgccag cctagccggg 6300 tcctcaacga caggagcacg atcatgcgca cccgtggccaggacccaacg ctgcccgaga 6360 tgcgccgcgt gcggctgctg gagatggcgg acgcgatggatatgttctgc caagggttgg 6420 tttgcgcatt cacagttctc cgcaagaatt gattggctccaattcttgga gtggtgaatc 6480 cgttagcgag gtgccgccgg cttccattca ggtcgaggtggcccggctcc atgcaccgcg 6540 acgcaacgcg gggaggcaga caaggtatag ggcggcgcctacaatccatg ccaacccgtt 6600 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatcagcggtccag tgatcgaagt 6660 taggctggta agagccgcga gcgatccttg aagctgtccctgatggtcgt catctacctg 6720 cctggacagc atggcctgca acgcgggcat cccgatgccgccggaagcga gaagaatcat 6780 aatggggaag gccatccagc ctcgcg 6806 24 3867 DNAArtificial Sequence Description of Artificial Sequence vector 24aagctttctc aagaagcgaa caagaaaaaa gaagagcaga ttaaacagct tcaagagttt 60gtcgctagat tcagcgccaa tgcgtctaaa tctaagcagg ctacatcaag aaagaaactt 120ctcgaaaaaa tcacgctgga tgatattaaa ccgtcttccc gccgctatcc ttatgttaac 180ttcacgccgg aacgggaaat cggaaatgat gttcttcgcg tggaaggctt aacaaaaaca 240atcgatggcg taaaggtgct tgacaatgtc agctttatta tgaatcgaga agataaaatt 300gctttcactg gccgaaatga acttgctgtt actacgctgt ttaaaatcat ttccggggaa 360atggaagctg acagcggaac gtttaaatgg ggtgttacca catctcaagc gtattttcca 420aaagacaaca gcgaatattt cgaaggcagt gatctgaacc ttgtagactg gcttcgccaa 480tattctccgc acgaccaaag tgagagcttt ttacgcggtt tcttaggacg catgctgttc 540tctggagaag aagtccacaa aaaagcaaat gtactttccg ggggagaaaa ggtccgctgt 600atgctgtcga aagcgatgct ttctggcgcc aatattttaa ttttggatga gccgaccaac 660catttagacc tagagtccat tacagcgctc aataacggct taatcagctt taaaggcgct 720atgctgttta cttcccatga ccatcagttt gtgcagacca ttgccaacag aattatagaa 780attacaccta acggcatcgt cgataagcaa atgagctatg acgagttcct tgaaaatgct 840gatgtgcaga aaaaattgac tgaactatac gccgaataaa aaagcagaga tttctctgct 900ttttttgata cctaaatgtg aaaggagatc acaacatgaa atttttggtt gtcggagcag 960gtggagtagg cgggtatatt ggcggacggc tttcggagaa aggaaatgat gtgacatttc 1020tcgtgcgcca aaaacgagct gagcagctta aaaaaaccgg gcttgtcatc catagtgaaa 1080aagggaatgt atcatttcag cccgaactaa tcagtgccgg agaaacaggg caatttgatg 1140tcgttatcat tgcttctaaa gcatactcgc ttggtcaagt gatagaggat gtcaaaccat 1200ttatccatca agaatctgtc attatccctt ttttaaatgg gtaccgccac tatgagcagc 1260tatttgcggc attttcaaaa gaacaggtgc tgggcggcct gtgttttata gaaagtgctt 1320tagacaacaa aggagaaatt catcatacga gcgcatcgca tcgttttgta tttggagaat 1380ggaacggcga gcgtacggag cggataagag cgcttgaaga ggcattttca ggtgtgaagg 1440ctgaagtcat cattagcggg catatcgaga agatcccctg cagcaatagt tacccttatt 1500atcaagataa gaaagaaaag gatttttcgc tacgctcaaa tcctttaaaa aaacacaaaa 1560gaccacattt tttaatgtgg tctttattct tcaactaaag cacccattag ttcaacaaac 1620gaaaattgga taaagtggga tatttttaaa atatatattt atgttacagt aatattgact 1680tttaaaaaag gattgattct aatgaagaaa gcagacaagt aagcctccta aattcacttt 1740agataaaaat ttaggaggca tatcaaatga actttaataa aattgattta gacaattgga 1800agagaaaaga gatatttaat cattatttga accaacaaac gacttttagt ataaccacag 1860aaattgatat tagtgtttta taccgaaaca taaaacaaga aggatataaa ttttaccctg 1920catttatttt cttagtgaca agggtgataa actcaaatac agcttttaga actggttaca 1980atagcgacgg agagttaggt tattgggata agttagagcc actttataca atttttgatg 2040gtgtatctaa aacattctct ggtatttgga ctcctgtaaa gaatgacttc aaagagtttt 2100atgatttata cctttctgat gtagagaaat ataatggttc ggggaaattg tttcccaaaa 2160cacctatacc tgaaaatgct ttttctcttt ctattattcc atggacttca tttactgggt 2220ttaacttaaa tatcaataat aatagtaatt accttctacc cattattaca gcaggaaaat 2280tcattaataa aggtaattca atatatttac cgctatcttt acaggtacat cattctgttt 2340gtgatggtta tcatgcagga ttgtttatga actctattca ggaattgtca gataggccta 2400atgactggct tttataatat gagataatgc cgactgtact ttttacagtc ggttttctaa 2460tgtcactaac ctgccccgtt agttgaagaa ggtttttata ttacagctcc cgggagatct 2520gggatcacta gtccaaacga cagaaggcga ccacctgcat ggatttttga ttgaaaaagc 2580aaaacgttta tctctcgctg caccagtatt agaaaccgtt tatgcgaatc tgcaaatgta 2640tgaagcagaa aaataaaaaa aggaggcgga aaagcctcct tttatttact taaaaagccc 2700aatttccgtt tctgaagata ggctctcttt tcccgtctgc cgtaattccg tcaatattca 2760tatccttaga accgatcata aagtccacgt gtgtaatgct ttcatttagg ccttctttga 2820caagctcttc acgagacatc tgctttccgc cttcaatatt aaaggcatag gcgcttccga 2880tcgccaaatg atttgacgcg ttttcatcaa acagcgtgtt atagaaaaga atgtttgatt 2940gtgatatagg cgaatcgtaa ggaacaagtg ccacttcacc taaatagtga gaaccttcat 3000ctgtttccac cagttctttt aaaatatcct cacctttttc agctttaatg tcgactatac 3060ggccattttc aaacgtcagg gtgaaatttt caataatatt tccgccgtag cttaatggtt 3120ttgtgcttga taccactccg tcaaccccgt ctttttgcgg cagcgtgaac acttcttctg 3180tcggcatatt ggccataaac tcatggccac tttcattcac gcttcccgca cctgcccaaa 3240catgcttcct aggcagctta attgttagat cagttccttc tgcttgataa tgtaaggcag 3300cgtaatgtct ctcgttcaaa tggtcaactt tttcatgaag attttggtca tgattgatcc 3360acgcctgaac cgggttgtct tcatttacgc gcgtcgcttt aaaaatttct tcccacagaa 3420ggtggatcgc ttcctcctct gatttgccag gaaacacctt gtgagcccag cctgctgatg 3480ccgcacctac gacagtccag ctgactttgt ctgattgaat atattgtctg tatgtatgta 3540atgctttgcc tgctgctttt tggaatgccg caatccgttt ggaatctata ccttttagca 3600agtctgggtt cgacgacaca acagaaatga aagcagctcc atttttggca agctcttctc 3660tgccttttgc ttcccattca ggatattctt caaatgcttc aaacggcgca agttcgtatt 3720ttaatttggc gacttcgtca tcctgccaat tcacggtgac gttttttgcg cccttttcat 3780atgcgtgttt tacaattaaa cggacaaaat cccgaacgtc tgttgaagca tttacgacta 3840catactggcc tttttggaca ttaacgc 3867 25 8704 DNA Artificial SequenceDescription of Artificial Sequence vector 25 gcggccgctt cgtcgaccgaaacagcagtt ataaggcatg aagctgtccg gtttttgcaa 60 aagtggctgt gactgtaaaaagaaatcgaa aaagaccgtt ttgtgtgaaa acggtctttt 120 tgtttccttt taaccaactgccataactcg aggcctacct agcttccaag aaagatatcc 180 taacagcaca agagcggaaagatgttttgt tctacatcca gaacaacctc tgctaaaatt 240 cctgaaaaat tttgcaaaaagttgttgact ttatctacaa ggtgtggtat aataatctta 300 acaacagcag gacgctctagaggaggagac taacatgaaa tttttggttg tcggagcagg 360 tggagtaggc gggtatattggcggacggct ttcggagaaa ggaaatgatg tgacatttct 420 cgtgcgccaa aaacgagctgagcagcttaa aaaaaccggg cttgtcatcc atagtgaaaa 480 agggaatgta tcatttcagcccgaactaat cagtgccgga gaaacagggc aatttgatgt 540 cgttatcatt gcttctaaagcatactcgct tggtcaagtg atagaggatg tcaaaccatt 600 tatccatcaa gaatctgtcattatcccttt tttaaatggg taccgccact atgagcagct 660 atttgcggca ttttcaaaagaacaggtgct gggcggcctg tgttttatag aaagtgcttt 720 agacaacaaa ggagaaattcatcatacgag cgcatcgcat cgttttgtat ttggagaatg 780 gaacggcgag cgtacggagcggataagagc gcttgaagag gcattttcag gtgtgaaggc 840 tgaagtcatc attagcgggcatatcgagaa ggacatttgg aaaaagtatc tctttattgc 900 agcgcaagcg gggatcacaacgttatttca acgaccgctt ggcccaatcc tcgccacaga 960 agccggacgt cacacggcccaaactcttat tggggaaatt tgcactgttt tacgaaaaga 1020 aggtgttccg gctgatccggctcttgagga agagagcttt cgtacgatga ccagcatgtc 1080 ttaccatatg aagtcctccatgcttcggga tatggaaaac ggccaaacga cagaaggcga 1140 ccacctgcat ggatttttgattgaaaaagc aaaacgttta tctctcgctg caccagtatt 1200 agaaaccgtt tatgcgaatctgcaaatgta tgaagcagaa aaataaaaaa aggaggcgga 1260 aaagcctcct tttatttacttaaaaagccc aatttccgtt tctgaagata ggctctcttt 1320 tcccgtctgc cgggatcctgttttggcgga tgagagaaga ttttcagcct gatacagatt 1380 aaatcagaac gcagaagcggtctgataaaa cagaatttgc ctggcggcag tagcgcggtg 1440 gtcccacctg accccatgccgaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg 1500 gggtctcccc atgcgagagtagggaactgc caggcatcaa ataaaacgaa aggctcagtc 1560 gaaagactgg gcctttcgttttatctgttg tttgtcggtg aacgctctcc tgagtaggac 1620 aaatccgccg ggagcggatttgaacgttgc gaagcaacgg cccggagggt ggcgggcagg 1680 acgcccgcca taaactgccaggcatcaaat taagcagaag gccatcctga cggatggcct 1740 ttttgcgttt ctacaaactcttggtaccca gaaaaagcgg caaaagcggc tgttaaaaaa 1800 gcgaaatcga agaagctgtctgccgctaag acggaatatc aaaagcgttc tgctgttgtg 1860 tcatctttaa aagtcacagccgatgaatcc cagcaagatg tcctaaaata cttgaacacc 1920 cagaaagata aaggaaatgcagaccaaatt cattcttatt atgtggtgaa cgggattgct 1980 gttcatgcct caaaagaggttatggaaaaa gtggtgcagt ttcccgaagt ggaaaaggtg 2040 cttcctaatg agaaacggcagctttttaag tcatcctccc catttaatat gaaaaaagca 2100 cagaaagcta ttaaagcaactgacggtgtg gaatggaatg tagaccaaat cgatgcccca 2160 aaagcttggg cacttggatatgatggaact ggcacggttg ttgcgtccat tgataccggg 2220 gtggaatgga atcatccggcattaaaagag aaatatcgcg gatataatcc ggaaaatcct 2280 aatgagcctg aaaatgaaatgaactggtat gatgccgtag caggcgaggc aagcccttat 2340 gatgatttgg ctcatggaacccacgtgaca ggcacgatgg tgggctctga acctgatgga 2400 acaaatcaaa tcggtgtagcacctggcgca aaatggattg ctgttaaagc gttctctgaa 2460 gatggcggca ctgatgctgacattttggaa gctggtgaat gggttttagc accaaaggac 2520 gcggaaggaa atccccacccggaaatggct cctgatgttg tcaataactc atggggaggg 2580 ggctctggac ttgatgaatggtacagagac atggtcaatg cctggcgttc ggccgatatt 2640 ttccctgagt tttcagcggggaatacggat ctctttattc ccggcgggcc tggttctatc 2700 gcaaatccgg caaactatccagaatcgttt gcaactggag cgactgagaa ttccaattcc 2760 ccatggagag aaaagaaaatcgctaatgtt gattactttg aacttctgca tattcttgaa 2820 tttaaaaagg ctgaaagagtaaaagattgt gctgaaatat tagagtataa acaaaatcgt 2880 gaaacaggcg aaagaaagttgtatcgagtg tggttttgta aatccaggct ttgtccaatg 2940 tgcaactgga ggagagcaatgaaacatggc attcagtcac aaaaggttgt tgctgaagtt 3000 attaaacaaa agccaacagttcgttggttg tttctcacat taacagttaa aaatgtttat 3060 gatggcgaag aattaaataagagtttgtca gatatggctc aaggatttcg ccgaatgatg 3120 caatataaaa aaattaataaaaatcttgtt ggttttatgc gtgcaacgga agtgacaata 3180 aataataaag ataattcttataatcagcac atgcatgtat tggtatgtgt ggaaccaact 3240 tattttaaga atacagaaaactacgtgaat caaaaacaat ggattcaatt ttggaaaaag 3300 gcaatgaaat tagactatgatccaaatgta aaagttcaaa tgattcgacc gaaaaataaa 3360 tataaatcgg atatacaatcggcaattgac gaaactgcaa aatatcctgt aaaggatacg 3420 gattttatga ccgatgatgaagaaaagaat ttgaaacgtt tgtctgattt ggaggaaggt 3480 ttacaccgta aaaggttaatctcctatggt ggtttgttaa aagaaataca taaaaaatta 3540 aaccttgatg acacagaagaaggcgatttg attcatacag atgatgacga aaaagccgat 3600 gaagatggat tttctattattgcaatgtgg aattgggaac ggaaaaatta ttttattaaa 3660 gagtagttca acaaacgggccatattgttg tataagtgat gaaatactga atttaaaact 3720 tagtttatat gtggtaaaatgttttaatca agtttaggag gaattaatta tgaagtgtaa 3780 tgaatgtaac agggttcaattaaaagaggg aagcgtatca ttaaccctat aaactacgtc 3840 tgccctcatt attggagggtgaaatgtgaa tacatcctat tcacaatcga atttacgaca 3900 caaccaaatt ttaatttggctttgcatttt atcttttttt agcgtattaa atgaaatggt 3960 tttgaacgtc tcattacctgatattgcaaa tgattttaat aaaccacctg cgagtacaaa 4020 ctgggtgaac acagcctttatgttaacctt ttccattgga acagctgtat atggaaagct 4080 atctgatcaa ttaggcatcaaaaggttact cctatttgga attataataa attgtttcgg 4140 gtcggtaatt gggtttgttggccattcttt cttttcctta cttattatgg ctcgttttat 4200 tcaaggggct ggtgcagctgcatttccagc actcgtaatg gttgtagttg cgcgctatat 4260 tccaaaggaa aataggggtaaagcatttgg tcttattgga tcgatagtag ccatgggaga 4320 aggagtcggt ccagcgattggtggaatgat agcccattat attcattggt cctatcttct 4380 actcattcct atgataacaattatcactgt tccgtttctt atgaaattat taaagaaaga 4440 agtaaggata aaaggtcattttgatatcaa aggaattata ctaatgtctg taggcattgt 4500 attttttatg ttgtttacaacatcatatag catttctttt cttatcgtta gcgtgctgtc 4560 attcctgata tttgtaaaacatatcaggaa agtaacagat ccttttgttg atcccggatt 4620 agggaaaaat ataccttttatgattggagt tctttgtggg ggaattatat ttggaacagt 4680 agcagggttt gtctctatggttccttatat gatgaaagat gttcaccagc taagtactgc 4740 cgaaatcgga agtgtaattattttccctgg aacaatgagt gtcattattt tcggctacat 4800 tggtgggata cttgttgatagaagaggtcc tttatacgtg ttaaacatcg gagttacatt 4860 tctttctgtt agctttttaactgcttcctt tcttttagaa acaacatcat ggttcatgac 4920 aattataatc gtatttgttttaggtgggct ttcgttcacc aaaacagtta tatcaacaat 4980 tgtttcaagt agcttgaaacagcaggaagc tggtgctgga atgagtttgc ttaactttac 5040 cagcttttta tcagagggaacaggtattgc aattgtaggt ggtttattat ccataccctt 5100 acttgatcaa aggttgttacctatggaagt tgatcagtca acttatctgt atagtaattt 5160 gttattactt ttttcaggaatcattgtcat tagttggctg gttaccttga atgtatataa 5220 acattctcaa agggatttctaaatcgttaa gggatcaact ttgggagaga gttcaaaatt 5280 gatccttttt ttataacagttcgaagcggc cgcaattctt gaagacgaaa gggcctcgtg 5340 atacgcctat ttttataggttaatgtcatg ataataatgg tttcttagac gtcaggtggc 5400 acttttcggg gaaatgtgcgcggaacccct atttgtttat ttttctaaat acattcaaat 5460 atgtatccgc tcatgagacaataaccctga taaatgcttc aataatattg aaaaaggaag 5520 agtatgagta ttcaacatttccgtgtcgcc cttattccct tttttgcggc attttgcctt 5580 cctgtttttg ctcacccagaaacgctggtg aaagtaaaag atgctgaaga tcagttgggt 5640 gcacgagtgg gttacatcgaactggatctc aacagcggta agatccttga gagttttcgc 5700 cccgaagaac gttttccaatgatgagcact tttaaagttc tgctatgtgg cgcggtatta 5760 tcccgtattg acgccgggcaagagcaactc ggtcgccgca tacactattc tcagaatgac 5820 ttggttgagt actcaccagtcacagaaaag catcttacgg atggcatgac agtaagagaa 5880 ttatgcagtg ctgccataaccatgagtgat aacactgcgg ccaacttact tctgacaacg 5940 atcggaggac cgaaggagctaaccgctttt ttgcacaaca tgggggatca tgtaactcgc 6000 cttgatcgtt gggaaccggagctgaatgaa gccataccaa acgacgagcg tgacaccacg 6060 atgcctgcag caatggcaacaacgttgcgc aaactattaa ctggcgaact acttactcta 6120 gcttcccggc aacaattaatagactggatg gaggcggata aagttgcagg accacttctg 6180 cgctcggccc ttccggctggctggtttatt gctgataaat ctggagccgg tgagcgtggg 6240 tctcgcggta tcattgcagcactggggcca gatggtaagc cctcccgtat cgtagttatc 6300 tacacgacgg ggagtcaggcaactatggat gaacgaaata gacagatcgc tgagataggt 6360 gcctcactga ttaagcattggtaactgtca gaccaagttt actcatatat actttagatt 6420 gatttaaaac ttcatttttaatttaaaagg atctaggtga agatcctttt tgataatctc 6480 atgaccaaaa tcccttaacgtgagttttcg ttccactgag cgtcagaccc cgtagaaaag 6540 atcaaaggat cttcttgagatccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 6600 aaaccaccgc taccagcggtggtttgtttg ccggatcaag agctaccaac tctttttccg 6660 aaggtaactg gcttcagcagagcgcagata ccaaatactg tccttctagt gtagccgtag 6720 ttaggccacc acttcaagaactctgtagca ccgcctacat acctcgctct gctaatcctg 6780 ttaccagtgg ctgctgccagtggcgataag tcgtgtctta ccgggttgga ctcaagacga 6840 tagttaccgg ataaggcgcagcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6900 ttggagcgaa cgacctacaccgaactgaga tacctacagc gtgagctatg agaaagcgcc 6960 acgcttcccg aagggagaaaggcggacagg tatccggtaa gcggcagggt cggaacagga 7020 gagcgcacga gggagcttccagggggaaac gcctggtatc tttatagtcc tgtcgggttt 7080 cgccacctct gacttgagcgtcgatttttg tgatgctcgt caggggggcg gagcctatgg 7140 aaaaacgcca gcaacgcggcctttttacgg ttcctggcct tttgctggcc ttttgctcac 7200 atgttctttc ctgcgttatcccctgattct gtggataacc gtattaccgc ctttgagtga 7260 gctgataccg ctcgccgcagccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 7320 gaagagcgcc tgatgcggtattttctcctt acgcatctgt gcggtatttc acaccgcata 7380 tggtgcactc tcagtacaatctgctctgat gccgcatagt taagccagta tacactccgc 7440 tatcgctacg tgactgggtcatggctgcgc cccgacaccc gccaacaccc gctgacgcgc 7500 cctgacgggc ttgtctgctcccggcatccg cttacagaca agctgtgacc gtctccggga 7560 gctgcatgtg tcagaggttttcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa 7620 gctcatcagc gtggtcgtgaagcgattcac agatgtctgc ctgttcatcc gcgtccagct 7680 cgttgagttt ctccagaagcgttaatgtct ggcttctgat aaagcgggcc atgttaaggg 7740 cggttttttc ctgtttggtcacttgatgcc tccgtgtaag ggggaatttc tgttcatggg 7800 ggtaatgata ccgatgaaacgagagaggat gctcacgata cgggttactg atgatgaaca 7860 tgcccggtta ctggaacgttgtgagggtaa acaactggcg gtatggatgc ggcgggacca 7920 gagaaaaatc actcagggtcaatgccagcg cttcgttaat acagatgtag gtgttccaca 7980 gggtagccag cagcatcctgcgatgcagat ccggaacata atggtgcagg gcgctgactt 8040 ccgcgtttcc agactttacgaaacacggaa accgaagacc attcatgttg ttgctcaggt 8100 cgcagacgtt ttgcagcagcagtcgcttca cgttcgctcg cgtatcggtg attcattctg 8160 ctaaccagta aggcaaccccgccagcctag ccgggtcctc aacgacagga gcacgatcat 8220 gcgcacccgt ggccaggacccaacgctgcc cgagatgcgc cgcgtgcggc tgctggagat 8280 ggcggacgcg atggatatgttctgccaagg gttggtttgc gcattcacag ttctccgcaa 8340 gaattgattg gctccaattcttggagtggt gaatccgtta gcgaggtgcc gccggcttcc 8400 attcaggtcg aggtggcccggctccatgca ccgcgacgca acgcggggag gcagacaagg 8460 tatagggcgg cgcctacaatccatgccaac ccgttccatg tgctcgccga ggcggcataa 8520 atcgccgtga cgatcagcggtccagtgatc gaagttaggc tggtaagagc cgcgagcgat 8580 ccttgaagct gtccctgatggtcgtcatct acctgcctgg acagcatggc ctgcaacgcg 8640 ggcatcccga tgccgccggaagcgagaaga atcataatgg ggaaggccat ccagcctcgc 8700 gtcg 8704

What is claimed:
 1. A process for the production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), comprisingculturing a microorganism under conditions such that HMBPA is producedand detecting the HMBPA produced by said microorganism.
 2. A process forthe production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid(HMBPA), comprising culturing a microorganism under conditions such thatHMBPA is produced and isolating the HMBPA produced by saidmicroorganism.
 3. A process for the production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), comprisingculturing a microorganism having increased keto reductase activity orincreased pantothenate synthetase activity in the presence of excessα-ketoisovalerate and excess β-alanine, such that HMBPA is produced. 4.A process for the production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), comprisingculturing a microorganism having increased keto reductase activity andincreased pantothenate synthetase activity in the presence of excessα-ketoisovalerate and excess β-alanine, such that HMBPA is produced. 5.The process of claim 3 or 4, wherein said microorganism comprises amodified panE gene.
 6. The process of claim 5, wherein the panE gene isoverexpressed, deregulated or present in multiple copies.
 7. The processof claim 3 or 4, wherein said microorganism comprises a modified panE1gene.
 8. The process of claim 3 or 4, wherein said microorganismcomprises a modified panE2 gene.
 9. The process of claim 3 or 4, whereinsaid microorganism comprises a modified panE1 gene and a modified panE2gene.
 10. The process of claim 3 or 4, wherein said microorganismcomprises a modified panC gene.
 11. The process of claim 3 or 4, whereinthe panC gene is overexpressed, deregulated or present in multiplecopies.
 12. The process of claim 3 or 4, wherein said microorganismfurther has increased acetohydroxyacid isomeroreductase activity.
 13. Aprocess for the production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), comprisingculturing a microorganism having increased acetohydroxyacidisomeroreductase activity in the presence of excess α-ketoisovalerateand excess β-alanine, such that HMBPA is produced.
 14. The process ofclaim 12 or 13, wherein said microorganism comprises a modified ilvCgene.
 15. The process of claim 14, wherein the ilvC gene isoverexpressed, deregulated or present in multiple copies.
 16. Theprocess of any one of claims 3, 4 or 11, wherein said microorganismfurther has reduced ketopantoate hydroxymethyltransferase activity. 17.The process of claim 16, wherein said microorganism comprises a modifiedpanB gene.
 18. The process of claim 16, wherein said microorganism hasbeen deleted for the panB gene.
 19. A process for the production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), comprisingculturing a microorganism having reduced ketopantoatehydroxymethyltransferase activity in the presence of excessα-ketoisovalerate and excess β-alanine, such that HMBPA is produced. 20.A method for enhancing production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) relative topantothenate, comprising culturing a recombinant microorganism underconditions such that the HMBPA production is enhanced relative topantothenate production.
 21. A process for the production of2-hydroxyisovaleric acid (α-HIV), comprising culturing a microorganismwhich overexpresses PanE1 or PanE2 and which further has reduced PanC orPanD activity under conditions such that α-HIV is produced.
 22. Aprocess for the production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), comprisingculturing a recombinant microorganism having decreased expression oractivity of serA or glyA under conditions such that HMBPA is produced.23. A process for the production of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), comprisingculturing a recombinant microorganism having decreased expression oractivity of serA and galA under conditions such that HMBPA is produced.24. The process of any one of the proceeding claims wherein themicroorganism is cultured under conditions of increased steady stateglucose.
 25. The process of any one of the proceeding claims wherein themicroorganism is cultured under conditions of decreased steady statedissolved oxygen.
 26. The process of any one of the proceeding claimswherein the microorganism is cultured under conditions of decreasedserine.
 27. A product produced according to any one of the above claims.28. A recombinant microorganism that produces3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), themicroorganism having a modification in at least one gene encodingketopantoate reductase that results in increased reductase activity andhaving a mutation or deletion in the panB gene that results in reducedketopantoate hydroxymethyltransferase activity.
 29. The recombinantmicroorganism of claim 28, wherein the gene encoding ketopantoatereductase is a panE gene.
 30. The recombinant microorganism of claim 29,wherein the panE gene is panE1.
 31. The recombinant microorganism ofclaim 29, wherein the panE gene is panE2.
 32. The recombinantmicroorganism of claim 28, wherein the microorganism has a modificationin panE1 and panE2.
 33. The recombinant microorganism of claim 28,further having a modification in ilvC that results in increasedacetohydroxyacid isomeroreductase activity.
 34. A recombinantmicroorganism that produces3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA), themicroorganism having a modification in ilvC that results in increasedacetohydroxyacid isomeroreductase activity and having a mutation ordeletion in the panB gene that results in reduced ketopantoatehydroxymethyltransferase activity.
 35. The recombinant microorganism ofany one of claims 28 to 34, wherein said microorganism belongs to thegenus Bacillus.
 36. The recombinant microorganism of claim 35, which isBacillus subtilis.